Targeted Therapeutic Nanoparticles And Methods Of Making And Using Same

ABSTRACT

Described herein are polymeric nanoparticles that include a therapeutic agent, and methods of making and using such therapeutic nanoparticles. In some embodiments, the contemplated nanoparticles may include an excipient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application No. 62/020,609, filed on Jul. 3, 2014, the entiretyof which is incorporated herein by reference.

BACKGROUND

Systems that deliver certain drugs to a patient (e.g., targeted to aparticular tissue or cell type or targeted to a specific diseased tissuebut not normal tissue) or that control release of drugs have long beenrecognized as beneficial.

For example, therapeutics that include an active drug and that are,e.g., targeted to a particular tissue or cell type or targeted to aspecific diseased tissue but not to normal tissue, may reduce the amountof the drug in tissues of the body that are not targeted. This isparticularly important when treating a condition such as cancer where itis desirable that a cytotoxic dose of the drug is delivered to cancercells without killing the surrounding non-cancerous tissue. Effectivedrug targeting may reduce the undesirable and sometimes life threateningside effects common in anticancer therapy. In addition, suchtherapeutics may allow drugs to reach certain tissues they wouldotherwise be unable to reach.

Therapeutics that offer controlled release and/or targeted therapy mustbe able to deliver an effective amount of drug, which is a knownlimitation in other nanoparticle delivery systems. For example, it canbe a challenge to prepare nanoparticle systems that have an appropriateamount of drug associated with each nanoparticle, while keeping the sizeof the nanoparticles small enough to have advantageous deliveryproperties. However, while it is desirable to load a nanoparticle with ahigh quantity of therapeutic agent, nanoparticle preparations that use adrug load that is too high will result in nanoparticles that are toolarge for practical therapeutic use.

Accordingly, a need exists for nanoparticle therapeutics and methods ofmaking such nanoparticles, that are capable of delivering therapeuticlevels of drug to treat diseases such as cancer, while also reducingpatient side effects.

SUMMARY

Described herein are polymeric nanoparticles that include a therapeuticagent, and methods of making and using such therapeutic nanoparticles.In some embodiments, the contemplated nanoparticles may include aexcipient.

In one aspect, a therapeutic nanoparticle is provided. The therapeuticnanoparticle comprises about 0.05 to about 30 weight percent of anexcipient selected from the group consisting of a polyanionic polymerand a polycationic polymer; about 0.2 to about 35 weight percent of atherapeutic agent; and about 35 to about 99.75 weight percent of abiocompatible polymer.

In certain embodiments, the excipient is a polyanionic polymer. Forexample, in certain embodiments, the polyanionic polymer is a copolymerof methacrylic acid and methyl methacrylate subunits. In some cases, theratio of methacrylic acid to methyl methacrylate subunits in thepolyanionic polymer is between about 1:0.9 to about 1:3.

In certain embodiments, the excipient is a polycationic polymer. Forexample, in certain embodiments, the polycationic polymer is a copolymerof alkyl methacrylate and dimethylaminoethylmethacrylate. In some cases,the polycationic polymer is a copolymer of dimethylaminoethylmethacrylate, butyl methacrylate, and methyl methacrylate subunits. Incertain embodiments, the ratio of dimethylaminoethyl methacrylate tobutyl methacrylate to methyl methacrylate subunits in the polycationicpolymer is about 1:2:1.

In certain embodiments, the excipient has a molecular weight of betweenabout 20 kDa and about 60 kDa, or between about 100 kDa and about 150kDa.

In certain embodiments, the excipient has a glass transition temperatureof between about 40° C. and about 50° C., or greater than about 100° C.

In certain embodiments, a contemplated nanoparticle comprises about 5 toabout 25 weight percent of the excipient.

In certain embodiments, a contemplated nanoparticle further comprisesabout 0.05 to about 35 weight percent cyclodextrin, or about 15 to about30 weight percent cyclodextrin. In some embodiments, the cyclodextrin isselected from the group consisting of α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, and mixtures thereof.

In certain embodiments, the therapeutic agent is a chemotherapeuticagent. For example, in certain embodiments the chemotherapeutic agent isselected from the group consisting of docetaxel, vincristine,vinorelbine, an epothilone, epothilone B, fluorouracil, irinotecan,capecitabine, and oxaliplatin. In certain embodiments, the therapeuticagent is a celecoxib. In certain embodiments, contemplated nanoparticlescomprise about 3 to about 20 weight percent of the therapeutic agent, orabout 5 to about 15 weight percent of the therapeutic agent.

In certain embodiments, the hydrodynamic diameter of a contemplatedtherapeutic nanoparticle is about 60 to about 200 nm, or about 90 toabout 140 nm.

In certain embodiments, the biocompatible polymer is selected from thegroup consisting of poly(lactic) acid-poly(ethylene)glycol copolymer andpoly(lactic) acid-co-poly(glycolic) acid-poly(ethylene)glycol copolymer.In certain embodiments, the poly(lactic) acid-poly(ethylene)glycolcopolymer has a poly(lactic) acid number average molecular weightfraction of about 0.6 to about 0.95, about 0.6 to about 0.8, about 0.75to about 0.85, or about 0.7 to about 0.9.

In certain embodiments, a contemplated therapeutic nanoparticlecomprises about 10 to about 25 weight percent poly(ethylene)glycol,about 10 to about 20 weight percent poly(ethylene)glycol, about 15 toabout 25 weight percent poly(ethylene)glycol, or about 20 to about 30weight percent poly(ethylene)glycol.

In certain embodiments, a contemplated therapeutic nanoparticle furthercomprises a1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene)glycolcopolymer.

In certain embodiments, a contemplated therapeutic nanoparticlesubstantially immediately releases less than about 70% of thetherapeutic agent after 0.5 hours when placed in a phosphate buffersolution at 37° C.

In another aspect, a therapeutic nanoparticle is provided. Thetherapeutic nanoparticle is prepared by a process comprisingemulsification of a first organic phase comprising a therapeutic agentor a pharmaceutically acceptable salt thereof, an excipient selectedfrom the group consisting of a polyanionic polymer and a polycationicpolymer, and a diblock poly(lactic)acid-polyethylene glycol or a diblockpoly(lactic)acid-co-poly(glycolic)acid-polyethylene glycol polymer, toform an emulsion phase; quenching the emulsion phase to form a quenchedphase; and filtration of the quenched phase to recover the therapeuticnanoparticle.

In yet another aspect, a pharmaceutically acceptable composition isprovided. The pharmaceutically acceptable composition comprises aplurality of contemplated therapeutic nanoparticles, and apharmaceutically acceptable excipient.

In certain embodiments, a contemplated pharmaceutically acceptablecomposition further comprises a saccharide and/or cyclodextrin. Forexample, in certain embodiments, the saccharide is a disaccharideselected from the group consisting of sucrose or trehalose, or a mixturethereof. In certain embodiments, the cyclodextrin is selected from thegroup consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, andmixtures thereof.

In still another aspect, a method of preparing a therapeuticnanoparticle is provided. The therapeutic nanoparticle comprisescombining a therapeutic agent and a first polymer with an organicsolvent to form a first organic phase having about 5 to about 50%solids; combining the first organic phase with a first aqueous solutionto form a second phase, wherein the first aqueous phase comprises anon-ionic surfactant; emulsifying the second phase to form an emulsionphase; quenching the emulsion phase to form a quenched phase; andfiltering the solubilized phase to recover the therapeuticnanoparticles, thereby forming a slurry of therapeutic nanoparticleshaving a diameter of about 80 nm to about 180 nm.

In certain embodiments, the non-ionic surfactant has ahydrophilic-lipophilic-balance (HLB) greater than about 15. For example,in certain embodiments, the non-ionic surfactant has ahydrophilic-lipophilic-balance (HLB) between about 15 and about 20. Incertain embodiments, the non-ionic surfactant is a polymeric non-ionicsurfactant.

In certain embodiments, the non-ionic surfactant comprises polyethyleneoxide or a copolymer thereof. For example, in certain embodiments, thepolymeric non-ionic surfactant is a copolymer non-ionic surfactantselected from poly(lactic acid)-polyethylene oxide copolymer andpoly(lactic acid)-co-(glycolic acid)-polyethylene oxide copolymer. Incertain embodiments, the copolymer non-ionic surfactant comprisespolyethylene oxide having a molecular weight between about 2 kDa andabout 10 kDa, or between about 4 kDa and about 6 kDa. In certainembodiments, the copolymer non-ionic surfactant comprises poly(lacticacid)-polyethylene oxide copolymer wherein the poly(lactic) acid has amolecular weight between about 0.2 kDa and about 1.0 kDa. In certainembodiments, the copolymer non-ionic surfactant comprises poly(lacticacid)-polyethylene oxide copolymer with the poly(lactic) acid having amolecular weight between about 0.4 kDa and about 0.8 kDa and thepolyethylene oxide having a molecular weight between about 4 kDa andabout 6 kDa.

In certain embodiments, the surfactant is a polyoxyethylene stearylether. For example, in certain embodiments, the polyoxyethylene stearylether is polyoxyethylene (100) stearyl ether. In certain embodiments,the polyoxyethylene stearyl ether has a molecular weight of between 4kDa and 6 kDa.

In certain embodiments, the aqueous solution comprises between about0.01 and about 5 weight percent of the surfactant, between about 0.01and about 1 weight percent of the surfactant, between about 0.05 andabout 0.2 weight percent of the surfactant, between about 1 and about 5weight percent of the surfactant, or between about 0.01 and about 5weight percent of the surfactant.

In certain embodiments, contemplated nanoparticles comprise about 0.2 toabout 35 weight percent of the therapeutic agent, about 3 to about 35weight percent of the therapeutic agent, or about 4 to about 15 weightpercent of the therapeutic agent. In certain embodiments, thetherapeutic agent is selected from the group consisting of docetaxel,vincristine, vinorelbine, an epothilone, epothilone B, fluorouracil,irinotecan, capecitabine, oxaliplatin, and celecoxib.

In certain embodiments, the first polymer comprises polylacticacid-polyethylene glycol diblock co-polymer. In certain embodiments, thefirst polymer comprises poly(lactide-co-glycolide)-poly(ethylene glycol)diblock copolymer.

In yet another aspect, a method of treating cancer in a patient in needthereof is provided. The method comprises administering to the patient atherapeutically effective amount of a composition comprising acontemplated therapeutic nanoparticle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart for an emulsion process for forming disclosednanoparticles, according to an embodiment.

FIGS. 2A and 2B are flow diagrams for a disclosed emulsion process. FIG.2A shows particle formation and hardening (upstream processing). FIG. 2Bshows particle work up and purification (downstream processing),according to an embodiment.

FIG. 3 shows in vitro release data for contemplated patupilonenanoparticle formulations containing a Eudragit excipient as compared toa control.

FIG. 4 shows in vitro release data for contemplated docetaxelnanoparticle formulations containing a Eudragit excipient and/orcyclodextrin as compared to a control.

FIG. 5 shows in vitro release data for contemplated celecoxibnanoparticle formulations containing a Eudragit excipient and/orcyclodextrin as compared to controls.

FIG. 6 shows in vitro release data for contemplated docetaxelnanoparticle formulations containing a polyethylene oxide-polylacticacid block copolymer (PEO-PEG) excipient as compared to controls.

FIG. 7 shows in vitro release data for contemplated docetaxelnanoparticle formulations containing a Brij® 100 excipient.

FIG. 8 shows in vitro release data for contemplated docetaxelnanoparticle formulations containing a polyethylene oxide-polylacticacid block copolymer excipient as compared to a control.

DETAILED DESCRIPTION

Described herein are polymeric nanoparticles that include a therapeuticagent, and methods of making and using such therapeutic nanoparticles.In some embodiments, the contemplated nanoparticles may include aexcipient. In some cases, including the excipient in a nanoparticleformulation enhances nanoparticle properties, such as the drug loadingof the nanoparticle, as compared to nanoparticles formulated without theexcipient.

Nanoparticles disclosed herein include one, two, three or morebiocompatible and/or biodegradable polymers. For example, a contemplatednanoparticle may include about 35 to about 99.6 weight percent, in someembodiments about 50 to about 99.6 weight percent, in some embodimentsabout 50 to about 99.5 weight percent, in some embodiments about 50 toabout 99 weight percent, in some embodiments about 50 to about 98 weightpercent, in some embodiments about 50 to about 97 weight percent, insome embodiments about 50 to about 96 weight percent, in someembodiments about 50 to about 95 weight percent, in some embodimentsabout 50 to about 94 weight percent, in some embodiments about 50 toabout 93 weight percent, in some embodiments about 50 to about 92 weightpercent, in some embodiments about 50 to about 91 weight percent, insome embodiments about 50 to about 90 weight percent, in someembodiments about 50 to about 85 weight percent, and in some embodimentsabout 50 to about 80 weight percent of one or more block copolymers thatinclude a biodegradable polymer and poly(ethylene glycol) (PEG), andabout 0 to about 50 weight percent of a biodegradable homopolymer.

In some embodiments, disclosed nanoparticles may include about 0.2 toabout 35 weight percent, about 0.2 to about 30 weight percent, about 0.2to about 20 weight percent, about 0.2 to about 10 weight percent, about0.2 to about 5 weight percent, about 0.5 to about 5 weight percent,about 0.75 to about 5 weight percent, about 1 to about 5 weight percent,about 2 to about 5 weight percent, about 3 to about 5 weight percent,about 1 to about 30 weight percent, about 1 to about 20 weight percent,about 2 to about 20 weight percent, about 5 to about 20 weight percent,about 1 to about 15 weight percent, about 2 to about 15 weight percent,about 3 to about 15 weight percent, about 4 to about 15 weight percent,about 5 to about 15 weight percent, about 1 to about 10 weight percent,about 2 to about 10 weight percent, about 3 to about 10 weight percent,about 4 to about 10 weight percent, about 5 to about 10 weight percent,about 10 to about 30 weight percent, or about 15 to about 25 weightpercent of an active agent.

In some embodiments, disclosed therapeutic nanoparticles may include anexcipient. An excipient may be any compound or mixture of compounds thatconfers an advantageous property to a nanoparticle. For example, in someembodiments, including an excipient in a nanoparticle formulation mayresult in increased drug loading in the nanoparticle as compared tonanoparticles formulated without the excipient. In another embodiment,the controlled release properties of a nanoparticle may beadvantageously altered by use of an excipient, e.g., the release ratemay be accelerated, slowed, etc. In certain embodiments, the size of thenanoparticles may be increased or decreased. In some cases, use of anexcipient may allow a nanoparticle to be formulated with less of orwithout one or more components yet have substantially similar propertiesas compared to nanoparticles formulated without the excipient, includingsubstantially similar particle size, drug loading, and/or release rate.

In some embodiments, the excipient may be a polymer. For example, insome instances, the excipient may be a polyanionic polymer or apolycationic polymer. Non-limiting examples of polyanionic andpolycationic polymers include, but are not limited to, polymers andcopolymers of acrylate and derivatives thereof (e.g., methacrylate andalkyl methacrylates such as methyl methacrylate, dimethylaminoethylmethacrylate, and butyl methacrylate). Non-limiting examples ofpolyanionic polymers include polyacrylic acid, polymethacrylic acid,Eudragit® S 100 (poly(methacrylic acid-co-methyl methacrylate) 1:2), andEudragit® L 100 (poly(methacrylic acid-co-methyl methacrylate) 1:1)).Non-limiting examples of polycationic polymers include Eudragit® E PO(poly(butyl methacrylate-co-(2-dimethylaminoethyl)methacrylate-co-methyl methacrylate) 1:2:1).

As discussed above, in some embodiments, the excipient may be acopolymer (e.g., formed from two or more polymer subunits). In someembodiments, a copolymer formed from a first subunit and a secondsubunit may have a ratio of the first subunit to the second subunit ofbetween about 5:1 to about 1:5, in some embodiments between about 4:1 toabout 1:4, in some embodiments between about 3:1 to about 1:3, in someembodiments between about 2:1 to about 1:2, in some embodiments betweenabout 1.5:1 to about 1:1.5, in some embodiments between about 2:1 toabout 1:5, in some embodiments between about 1:1 to about 1:5, in someembodiments between about 1:1 to about 1:4, in some embodiments betweenabout 1:1 to about 1:3, and in some embodiments between about 1:0.9 toabout 1:3.

In some embodiments, the excipient may have a molecular weight ofbetween about 20 kDa and about 200 kDa, in some embodiments betweenabout 20 kDa and about 150 kDa, in some embodiments between about 20 kDaand about 125 kDa, in some embodiments between about 20 kDa and about100 kDa, in some embodiments between about 20 kDa and about 75 kDa, insome embodiments between about 20 kDa and about 60 kDa, in someembodiments between about 40 kDa and about 60 kDa, in some embodimentsbetween about 50 kDa and about 200 kDa, in some embodiments betweenabout 75 kDa and about 200 kDa, in some embodiments between about 100kDa and about 200 kDa, in some embodiments between about 125 kDa andabout 200 kDa, and in some embodiments between about 100 kDa and about150 kDa.

In some embodiments, the excipient may have a glass transitiontemperature of between about 30° C. and about 130° C., in someembodiments between about 30° C. and about 100° C., in some embodimentsbetween about 30° C. and about 80° C., in some embodiments between about30° C. and about 60° C., in some embodiments between about 35° C. andabout 60° C., in some embodiments between about 40° C. and about 60° C.,or in some embodiments between about 40° C. and about 50° C. In certainembodiments, the excipient may have a glass transition temperature ofgreater than about 40° C., in some embodiments greater than about 50°C., in some embodiments greater than about 60° C., in some embodimentsgreater than about 80° C., in some embodiments greater than about 100°C., in some embodiments greater than about 120° C., or in someembodiments greater than about 130° C.

In some embodiments, a nanoparticle may comprise about 0.05 to about 35weight percent of the excipient, in some embodiments about 0.05 to about30 weight percent of the excipient, in some embodiments about 0.1 toabout 30 weight percent of the excipient, in some embodiments about 0.5to about 30 weight percent of the excipient, in some embodiments about 1to about 30 weight percent of the excipient, in some embodiments about 2to about 30 weight percent of the excipient, in some embodiments about 5to about 30 weight percent of the excipient, in some embodiments about10 to about 30 weight percent of the excipient, in some embodimentsabout 15 to about 30 weight percent of the excipient, in someembodiments about 20 to about 30 weight percent of the excipient, insome embodiments about 15 to about 25 weight percent of the excipient,in some embodiments about 5 to about 25 weight percent of the excipient,in some embodiments about 5 to about 20 weight percent of the excipient,or in some embodiments about 5 to about 15 weight percent of theexcipient.

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm, e.g., about 10 nm to about 200 nm. Disclosedtherapeutic nanoparticles may include nanoparticles having a diameter ofabout 60 to about 200 nm, about 60 to about 190 nm, or about 70 to about190 nm, or about 60 to about 180 nm, or about 70 nm to about 180 nm, orabout 50 nm to about 200 nm, or about 60 to about 120 nm, or about 70 toabout 120 nm, or about 80 to about 120 nm, or about 90 to about 120 nm,or about 100 to about 120 nm, or about 60 to about 130 nm, or about 70to about 130 nm, or about 80 to about 130 nm, or about 90 to about 130nm, or about 100 to about 130 nm, or about 110 to about 130 nm, or about60 to about 140 nm, or about 70 to about 140 nm, or about 80 to about140 nm, or about 90 to about 140 nm, or about 100 to about 140 nm, orabout 110 to about 140 nm, or about 60 to about 150 nm, or about 70 toabout 150 nm, or about 80 to about 150 nm, or about 90 to about 150 nm,or about 100 to about 150 nm, or about 110 to about 150 nm, or about 120to about 150 nm.

Polymers

In some embodiments, the nanoparticles may comprise a matrix of polymersand a therapeutic agent. In some embodiments, a therapeutic agent and/ortargeting moiety can be associated with at least part of the polymericmatrix. The therapeutic agent can be associated with the surface of,encapsulated within, surrounded by, and/or dispersed throughout thepolymeric matrix.

Any suitable polymer can be used in the disclosed nanoparticles.Polymers can be natural or unnatural (synthetic) polymers. Polymers canbe homopolymers or copolymers comprising two or more monomers. In termsof sequence, copolymers can be random, block, or comprise a combinationof random and block sequences. Typically, polymers are organic polymers.

The term “polymer,” as used herein, is given its ordinary meaning asused in the art, i.e., a molecular structure comprising one or morerepeat units (monomers), connected by covalent bonds. The repeat unitsmay all be identical, or in some cases, there may be more than one typeof repeat unit present within the polymer. In some cases, the polymercan be biologically derived, i.e., a biopolymer. Non-limiting examplesinclude peptides or proteins. In some cases, additional moieties mayalso be present in the polymer, for example biological moieties such asthose described below. If more than one type of repeat unit is presentwithin the polymer, then the polymer is said to be a “copolymer.” It isto be understood that in any embodiment employing a polymer, the polymerbeing employed may be a copolymer in some cases. The repeat unitsforming the copolymer may be arranged in any fashion. For example, therepeat units may be arranged in a random order, in an alternating order,or as a block copolymer, i.e., comprising one or more regions eachcomprising a first repeat unit (e.g., a first block), and one or moreregions each comprising a second repeat unit (e.g., a second block),etc. Block copolymers may have two (a diblock copolymer), three (atriblock copolymer), or more numbers of distinct blocks.

Disclosed particles can include copolymers, which, in some embodiments,describes two or more polymers (such as those described herein) thathave been associated with each other, usually by covalent bonding of thetwo or more polymers together. Thus, a copolymer may comprise a firstpolymer and a second polymer, which have been conjugated together toform a block copolymer where the first polymer can be a first block ofthe block copolymer and the second polymer can be a second block of theblock copolymer. Of course, those of ordinary skill in the art willunderstand that a block copolymer may, in some cases, contain multipleblocks of polymer, and that a “block copolymer,” as used herein, is notlimited to only block copolymers having only a single first block and asingle second block. For instance, a block copolymer may comprise afirst block comprising a first polymer, a second block comprising asecond polymer, and a third block comprising a third polymer or thefirst polymer, etc. In some cases, block copolymers can contain anynumber of first blocks of a first polymer and second blocks of a secondpolymer (and in certain cases, third blocks, fourth blocks, etc.). Inaddition, it should be noted that block copolymers can also be formed,in some instances, from other block copolymers. For example, a firstblock copolymer may be conjugated to another polymer (which may be ahomopolymer, a biopolymer, another block copolymer, etc.), to form a newblock copolymer containing multiple types of blocks, and/or to othermoieties (e.g., to non-polymeric moieties).

In some embodiments, the polymer (e.g., copolymer, e.g., blockcopolymer) can be amphiphilic, i.e., having a hydrophilic portion and ahydrophobic portion, or a relatively hydrophilic portion and arelatively hydrophobic portion. A hydrophilic polymer can be onegenerally that attracts water and a hydrophobic polymer can be one thatgenerally repels water. A hydrophilic or a hydrophobic polymer can beidentified, for example, by preparing a sample of the polymer andmeasuring its contact angle with water (typically, the polymer will havea contact angle of less than 60°, while a hydrophobic polymer will havea contact angle of greater than about 60°). In some cases, thehydrophilicity of two or more polymers may be measured relative to eachother, i.e., a first polymer may be more hydrophilic than a secondpolymer. For instance, the first polymer may have a smaller contactangle than the second polymer.

In one set of embodiments, a polymer (e.g., copolymer, e.g., blockcopolymer) contemplated herein includes a biocompatible polymer, i.e.,the polymer that does not typically induce an adverse response wheninserted or injected into a living subject, for example, withoutsignificant inflammation and/or acute rejection of the polymer by theimmune system, for instance, via a T-cell response. Accordingly, thetherapeutic particles contemplated herein can be non-immunogenic. Theterm non-immunogenic as used herein refers to endogenous growth factorin its native state which normally elicits no, or only minimal levelsof, circulating antibodies, T-cells, or reactive immune cells, and whichnormally does not elicit in the individual an immune response againstitself.

Biocompatibility typically refers to the acute rejection of material byat least a portion of the immune system, i.e., a nonbiocompatiblematerial implanted into a subject provokes an immune response in thesubject that can be severe enough such that the rejection of thematerial by the immune system cannot be adequately controlled, and oftenis of a degree such that the material must be removed from the subject.One simple test to determine biocompatibility can be to expose a polymerto cells in vitro; biocompatible polymers are polymers that typicallywill not result in significant cell death at moderate concentrations,e.g., at concentrations of 50 micrograms/10⁶ cells. For instance, abiocompatible polymer may cause less than about 20% cell death whenexposed to cells such as fibroblasts or epithelial cells, even ifphagocytosed or otherwise uptaken by such cells. Non-limiting examplesof biocompatible polymers that may be useful in various embodimentsinclude polydioxanone (PDO), polyhydroxyalkanoate, polyhydroxybutyrate,poly(glycerol sebacate), polyglycolide (i.e., poly(glycolic) acid)(PGA), polylactide (i.e., poly(lactic) acid) (PLA), poly(lactic)acid-co-poly(glycolic) acid (PLGA), polycaprolactone, or copolymers orderivatives including these and/or other polymers.

In certain embodiments, contemplated biocompatible polymers may bebiodegradable, i.e., the polymer is able to degrade, chemically and/orbiologically, within a physiological environment, such as within thebody. As used herein, “biodegradable” polymers are those that, whenintroduced into cells, are broken down by the cellular machinery(biologically degradable) and/or by a chemical process, such ashydrolysis, (chemically degradable) into components that the cells caneither reuse or dispose of without significant toxic effect on thecells. In one embodiment, the biodegradable polymer and theirdegradation byproducts can be biocompatible.

Particles disclosed herein may or may not contain PEG. In addition,certain embodiments can be directed towards copolymers containingpoly(ester-ether)s, e.g., polymers having repeat units joined by esterbonds (e.g., R—C(O)—O—R′ bonds) and ether bonds (e.g., R—O—R′ bonds). Insome embodiments, a biodegradable polymer, such as a hydrolyzablepolymer, containing carboxylic acid groups, may be conjugated withpoly(ethylene glycol) repeat units to form a poly(ester-ether). Apolymer (e.g., copolymer, e.g., block copolymer) containingpoly(ethylene glycol) repeat units can also be referred to as a“PEGylated” polymer.

For instance, a contemplated polymer may be one that hydrolyzesspontaneously upon exposure to water (e.g., within a subject), thepolymer may degrade upon exposure to heat (e.g., at temperatures ofabout 37° C.). Degradation of a polymer may occur at varying rates,depending on the polymer or copolymer used. For example, the half-lifeof the polymer (the time at which 50% of the polymer can be degradedinto monomers and/or other nonpolymeric moieties) may be on the order ofdays, weeks, months, or years, depending on the polymer. The polymersmay be biologically degraded, e.g., by enzymatic activity or cellularmachinery, in some cases, for example, through exposure to a lysozyme(e.g., having relatively low pH). In some cases, the polymers may bebroken down into monomers and/or other nonpolymeric moieties that cellscan either reuse or dispose of without significant toxic effect on thecells (for example, polylactide may be hydrolyzed to form lactic acid,polyglycolide may be hydrolyzed to form glycolic acid, etc.).

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEGylated polymers andcopolymers of lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA,PEGylated PLGA, and derivatives thereof). In some embodiments,polyesters include, for example, polyanhydrides, poly(ortho ester)PEGylated poly(ortho ester), poly(caprolactone), PEGylatedpoly(caprolactone), polylysine, PEGylated polylysine, poly(ethyleneimine), PEGylated poly(ethylene imine), poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA can be characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid-glycolic acid ratio. In some embodiments, PLGA can becharacterized by a lactic acid:glycolic acid ratio of approximately85:15, approximately 75:25, approximately 60:40, approximately 50:50,approximately 40:60, approximately 25:75, or approximately 15:85. Insome embodiments, the ratio of lactic acid to glycolic acid monomers inthe polymer of the particle (e.g., the PLGA block copolymer or PLGA-PEGblock copolymer), may be selected to optimize for various parameterssuch as water uptake, therapeutic agent release and/or polymerdegradation kinetics can be optimized.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid polyacrylamide, amino alkyl methacrylatecopolymer, glycidyl methacrylate copolymers, polycyanoacrylates, andcombinations comprising one or more of the foregoing polymers. Theacrylic polymer may comprise fully-polymerized copolymers of acrylic andmethacrylic acid esters with a low content of quaternary ammoniumgroups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g., DNA, RNA, or derivatives thereof).Amine-containing polymers such as poly(lysine), polyethylene imine(PEI), and poly(amidoamine) dendrimers are contemplated for use, in someembodiments, in a disclosed particle.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains. Examples of these polyesters includepoly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester).

It is contemplated that PEG may be terminated and include an end group.For example, PEG may terminate in a hydroxyl, a methoxy or other alkoxylgroup, a methyl or other alkyl group, an aryl group, a carboxylic acid,an amine, an amide, an acetyl group, a guanidino group, or an imidazole.Other contemplated end groups include azide, alkyne, maleimide,aldehyde, hydrazide, hydroxylamine, alkoxyamine, or thiol moieties.

Those of ordinary skill in the art will know of methods and techniquesfor PEGylating a polymer, for example, by using EDC(l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS(N-hydroxysuccinimide) to react a polymer to a PEG group terminating inan amine, by ring opening polymerization techniques (ROMP), or the like.

In one embodiment, the molecular weight (or e.g., the ratio of molecularweights of, e.g., different blocks of a copolymer) of the polymers canbe optimized for effective treatment as disclosed herein. For example,the molecular weight of a polymer may influence particle degradationrate (such as when the molecular weight of a biodegradable polymer canbe adjusted), solubility, water uptake, and drug release kinetics. Forexample, the molecular weight of the polymer (or e.g., the ratio ofmolecular weights of, e.g., different blocks of a copolymer) can beadjusted such that the particle biodegrades in the subject being treatedwithin a reasonable period of time (ranging from a few hours to 1-2weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks, etc.).

A disclosed particle can for example comprise a diblock copolymer of PEGand PL(G)A, wherein for example, the PEG portion may have a numberaverage molecular weight of about 1,000-20,000, e.g., about2,000-20,000, e.g., about 2 to about 10,000, and the PL(G)A portion mayhave a number average molecular weight of about 5,000 to about 20,000,or about 5,000-100,000, e.g., about 20,000-70,000, e.g., about15,000-50,000.

For example, disclosed here is an exemplary therapeutic nanoparticlethat includes about 10 to about 99 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly (glycolic)acid-poly(ethylene)glycol copolymer, or about 20 to about 80 weightpercent, about 40 to about 80 weight percent, or about 30 to about 50weight percent, or about 70 to about 90 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly (glycolic)acid-poly(ethylene)glycol copolymer. Exemplary poly(lactic)acid-poly(ethylene)glycol copolymers can include a number averagemolecular weight of about 15 to about 20 kDa (e.g. 15 or 16 kDa), orabout 10 to about 25 kDa of poly(lactic) acid and a number averagemolecular weight of about 4 to about 6 (e.g. 5 kDa), or about 2 kDa toabout 10 kDa of poly(ethylene)glycol.

In some embodiments, the poly(lactic) acid-poly(ethylene)glycolcopolymer may have a poly(lactic) acid number average molecular weightfraction of about 0.6 to about 0.95, in some embodiments between about0.7 to about 0.9, in some embodiments between about 0.6 to about 0.8, insome embodiments between about 0.7 to about 0.8, in some embodimentsbetween about 0.75 to about 0.85, in some embodiments between about 0.8to about 0.9, and in some embodiments between about 0.85 to about 0.95.It should be understood that the poly(lactic) acid number averagemolecular weight fraction may be calculated by dividing the numberaverage molecular weight of the poly(lactic) acid component of thecopolymer by the sum of the number average molecular weight of thepoly(lactic) acid component and the number average molecular weight ofthe poly(ethylene)glycol component.

Disclosed nanoparticles may optionally include about 1 to about 50weight percent poly(lactic) acid or poly(lactic) acid-co-poly (glycolic)acid (which does not include PEG), or may optionally include about 1 toabout 50 weight percent, or about 10 to about 50 weight percent or about30 to about 50 weight percent poly(lactic) acid or poly(lactic)acid-co-poly (glycolic) acid. For example, poly(lactic) orpoly(lactic)-co-poly(glycolic) acid may have a number average moleculeweight of about 5 to about 15 kDa, or about 5 to about 12 kDa. ExemplaryPLA may have a number average molecular weight of about 5 to about 10kDa. Exemplary PLGA may have a number average molecular weight of about8 to about 12 kDa.

A therapeutic nanoparticle may, in some embodiments, contain about 10 toabout 30 weight percent, in some embodiments about 10 to about 25 weightpercent, in some embodiments about 10 to about 20 weight percent, insome embodiments about 10 to about 15 weight percent, in someembodiments about 15 to about 20 weight percent, in some embodimentsabout 15 to about 25 weight percent, in some embodiments about 20 toabout 25 weight percent, in some embodiments about 20 to about 30 weightpercent, or in some embodiments about 25 to about 30 weight percent ofpoly(ethylene)glycol, where the poly(ethylene)glycol may be present as apoly(lactic) acid-poly(ethylene)glycol copolymer, poly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer, or poly(ethylene)glycolhomopolymer. In certain embodiments, the polymers of the nanoparticlescan be conjugated to a lipid. The polymer can be, for example, alipid-terminated PEG. As described below, the lipid portion of thepolymer can be used for self-assembly with another polymer, facilitatingthe formation of a nanoparticle. For example, a hydrophilic polymercould be conjugated to a lipid that will self-assemble with ahydrophobic polymer.

In some embodiments, lipids are oils. In general, any oil known in theart can be conjugated to the polymers used in the nanoparticles. In someembodiments, an oil can comprise one or more fatty acid groups or saltsthereof. In some embodiments, a fatty acid group can comprisedigestible, long chain (e.g., C₆-C₅₀), substituted or unsubstitutedhydrocarbons. In some embodiments, a fatty acid group can be a C₁₀-C₂₀fatty acid or salt thereof. In some embodiments, a fatty acid group canbe a C₁₅-C₂₀ fatty acid or salt thereof. In some embodiments, a fattyacid can be unsaturated. In some embodiments, a fatty acid group can bemonounsaturated. In some embodiments, a fatty acid group can bepolyunsaturated. In some embodiments, a double bond of an unsaturatedfatty acid group can be in the cis conformation. In some embodiments, adouble bond of an unsaturated fatty acid can be in the transconformation.

In some embodiments, a fatty acid group can be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group can be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In a particular embodiment, the lipid is of the Formula V:

and salts thereof, wherein each R is, independently, C₁₋₃₀ alkyl. In oneembodiment of Formula V, the lipid is 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof,e.g., the sodium salt.

In one embodiment, optional small molecule targeting moieties arebonded, e.g., covalently bonded, to the lipid component of thenanoparticle.

Nanoparticles

In one set of embodiments, the particles can have an interior and asurface, where the surface has a composition different from theinterior, i.e., there may be at least one compound present in theinterior but not present on the surface (or vice versa), and/or at leastone compound is present in the interior and on the surface at differingconcentrations. For example, in one embodiment, a compound, such as atargeting moiety, may be present in both the interior and the surface ofthe particle, but at a higher concentration on the surface than in theinterior of the particle. Although in some cases, the concentration inthe interior of the particle may be essentially nonzero, i.e., there isa detectable amount of the compound present in the interior of theparticle.

In some cases, the interior of the particle is more hydrophobic than thesurface of the particle. For instance, the interior of the particle maybe relatively hydrophobic with respect to the surface of the particle,and a drug or other payload may be hydrophobic, and readily associateswith the relatively hydrophobic center of the particle. The drug orother payload can thus be contained within the interior of the particle,which can shelter it from the external environment surrounding theparticle (or vice versa). For instance, a drug or other payloadcontained within a particle administered to a subject will be protectedfrom a subject's body, and the body may also be substantially isolatedfrom the drug for at least a period of time.

For example, disclosed herein is a therapeutic polymeric nanoparticlecomprising a first non-functionalized polymer; an optional secondnon-functionalized polymer; an optional functionalized polymercomprising a targeting moiety; and a therapeutic agent. In a particularembodiment, the first non-functionalized polymer is PLA, PLGA, or PEG,or copolymers thereof, e.g., a diblock co-polymer PLA-PEG. For example,exemplary nanoparticles may have a PEG corona with a density of about0.065 g/cm³, or about 0.01 to about 0.10 g/cm³.

Disclosed nanoparticles may be stable (e.g., retain substantially allactive agent) for example in a solution that may contain a saccharide,for at least about 3 days, about 4 days or at least about 5 days at roomtemperature, or at 25° C.

In some embodiments, a contemplated nanoparticle may comprise acyclodextrin. A suitable cyclodextrin may include α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, or mixtures thereof. Exemplarycyclodextrins contemplated for use in the nanoparticles disclosed hereininclude hydroxypropyl-β-cyclodextrin (HPbCD),hydroxyethyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin,methyl-β-cyclodextrin, cyclodextrin, carboxymethyl-β-cyclodextrin,carboxymethyl ethyl-β-cyclodextrin, diethyl-β-cyclodextrin,tri-O-alkyl-β-cyclodextrin, glucosyl-β-cyclodextrin, andmaltosyl-β-cyclodextrin. In some embodiments, the cyclodextrin may becovalently attached to polymer. For example, in some embodiments, thecyclodextrin may be covalently attached to chitosan.

For example, in some embodiments, a contemplated nanoparticle maycomprise about 0.05 to about 35 weight percent of a cyclodextrin, insome embodiments about 0.05 to about 30 weight percent of acyclodextrin, in some embodiments about 0.1 to about 30 weight percentof a cyclodextrin, in some embodiments about 0.5 to about 30 weightpercent of a cyclodextrin, in some embodiments about 1 to about 30weight percent of a cyclodextrin, in some embodiments about 2 to about30 weight percent of a cyclodextrin, in some embodiments about 5 toabout 30 weight percent of a cyclodextrin, in some embodiments about 10to about 30 weight percent of a cyclodextrin, in some embodiments about15 to about 30 weight percent of a cyclodextrin, in some embodimentsabout 20 to about 30 weight percent of a cyclodextrin, in someembodiments about 15 to about 25 weight percent of a cyclodextrin, insome embodiments about 5 to about 25 weight percent of a cyclodextrin,in some embodiments about 5 to about 20 weight percent of acyclodextrin, or in some embodiments about 5 to about 15 weight percentof a cyclodextrin.

In some embodiments, disclosed nanoparticles may also include a fattyalcohol, which may increase the rate of drug release. For example,disclosed nanoparticles may include a C₈-C₃₀ alcohol such as cetylalcohol, octanol, stearyl alcohol, arachidyl alcohol, docosonal, oroctasonal.

Nanoparticles may have controlled release properties, e.g., may becapable of delivering an amount of a therapeutic agent to a patient,e.g., to specific site in a patient, over an extended period of time,e.g., over 1 day, 1 week, or more.

In some embodiments, disclosed nanoparticles substantially immediatelyrelease (e.g., over about 1 minute to about 30 minutes) less than about2%, less than about 4%, less than about 5%, or less than about 10% of anactive agent, for example when placed in a phosphate buffer solution atroom temperature and/or at 37° C.

In another embodiment, a disclosed nanoparticle may release less thanabout 40%, less than 50%, or less than 60%, less than 70% of an activeagent for example when placed in a phosphate buffer solution at roomtemperature or at 37° C., for 0.5 hour or more. In one embodiment, adisclosed nanoparticle may release less than about 70% of thetherapeutic agent over 0.5 hour when placed in a phosphate buffersolution at 37° C.

In another embodiment, a disclosed nanoparticle may release less thanabout 20%, less than about 30%, less than about 40%, less than 50%, oreven less than 60% (or more) for example when placed in a phosphatebuffer solution at room temperature or at 37° C., for 1 day or more. Inone embodiment, a disclosed nanoparticle may release less than about 60%of the therapeutic agent over 2 hours when placed in a phosphate buffersolution at room temperature.

In some embodiments, after administration to a subject or patient of adisclosed nanoparticle or a composition that includes a disclosednanoparticle, the peak plasma concentration (C_(max)) of the therapeuticagent in the patient is substantially higher as compared to a C_(max) ofthe therapeutic agent if administered alone (e.g., not as part of ananoparticle).

In another embodiment, a disclosed nanoparticle including a therapeuticagent, when administered to a subject, may have a t_(max) of therapeuticagent substantially longer as compared to a t_(max) of the therapeuticagent administered alone.

Libraries of such particles may also be formed. For example, by varyingthe ratios of the two (or more) polymers within the particle, theselibraries can be useful for screening tests, high-throughput assays, orthe like. Entities within the library may vary by properties such asthose described above, and in some cases, more than one property of theparticles may be varied within the library. Accordingly, one embodimentis directed to a library of nanoparticles having different ratios ofpolymers with differing properties. The library may include any suitableratio(s) of the polymers.

In some embodiments, the biocompatible polymer is a hydrophobic polymer.Non-limiting examples of biocompatible polymers include polylactide,polyglycolide, and/or poly(lactide-co-glycolide).

In a different embodiment, this disclosure provides for a nanoparticlecomprising 1) a polymeric matrix; 2) optionally, an amphiphilic compoundor layer that surrounds or is dispersed within the polymeric matrixforming a continuous or discontinuous shell for the particle; 3) anon-functionalized polymer that may form part of the polymeric matrix,and 4) optionally, an excipient, which may form part of the polymericmatrix. For example, an amphiphilic layer may reduce water penetrationinto the nanoparticle, thereby enhancing drug encapsulation efficiencyand slowing drug release.

As used herein, the term “amphiphilic” refers to a property where amolecule has both a polar portion and a non-polar portion. Often, anamphiphilic compound has a polar head attached to a long hydrophobictail. In some embodiments, the polar portion is soluble in water, whilethe non-polar portion is insoluble in water. In addition, the polarportion may have either a formal positive charge, or a formal negativecharge. Alternatively, the polar portion may have both a formal positiveand a negative charge, and be a zwitterion or inner salt. In someembodiments, the amphiphilic compound can be, but is not limited to, oneor a plurality of the following: naturally derived lipids, surfactants,or synthesized compounds with both hydrophilic and hydrophobic moieties.

Specific examples of amphiphilic compounds include, but are not limitedto, phospholipids, such as 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), diarachidoylphosphatidylcholine (DAPC),dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine(DTPC), and dilignoceroylphatidylcholine (DLPC), incorporated at a ratioof between 0.01-60 (weight lipid/w polymer), most preferably between0.1-30 (weight lipid/w polymer). Phospholipids which may be usedinclude, but are not limited to, phosphatidic acids, phosphatidylcholines with both saturated and unsaturated lipids, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines,phosphatidylinositols, lysophosphatidyl derivatives, cardiolipin, andβ-acyl-y-alkyl phospholipids. Examples of phospholipids include, but arenot limited to, phosphatidylcholines such asdioleoylphosphatidylcholine, dimyristoylphosphatidylcholine,dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine,dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), diarachidoylphosphatidylcholine (DAPC),dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine(DTPC), dilignoceroylphatidylcholine (DLPC); andphosphatidylethanolamines such as dioleoylphosphatidylethanolamine or1-hexadecyl-2-palmitoylglycerophosphoethanolamine. Syntheticphospholipids with asymmetric acyl chains (e.g., with one acyl chain of6 carbons and another acyl chain of 12 carbons) may also be used.

In a particular embodiment, an amphiphilic component that can be used toform an amphiphilic layer is lecithin, and, in particular,phosphatidylcholine. Lecithin is an amphiphilic lipid and, as such,forms a phospholipid bilayer having the hydrophilic (polar) heads facingtheir surroundings, which are oftentimes aqueous, and the hydrophobictails facing each other. Lecithin has an advantage of being a naturallipid that is available from, e.g., soybean, and already has FDAapproval for use in other delivery devices. In addition, a mixture oflipids such as lethicin is more advantageous than one single pure lipid.

In certain embodiments a disclosed nanoparticle has an amphiphilicmonolayer, meaning the layer is not a phospholipid bilayer, but existsas a single continuous or discontinuous layer around, or within, thenanoparticle. The amphiphilic layer is “associated with” thenanoparticle, meaning it is positioned in some proximity to thepolymeric matrix, such as surrounding the outside of the polymericshell, or dispersed within the polymers that make up the nanoparticle.

Preparation of Nanoparticles

Another aspect of this disclosure is directed to systems and methods ofmaking disclosed nanoparticles. In some embodiments, using two or moredifferent polymers (e.g., copolymers, e.g., block copolymers) indifferent ratios and producing particles from the polymers (e.g.,copolymers, e.g., block copolymers), properties of the particles becontrolled. For example, a polymer (e.g., copolymer, e.g., blockcopolymer) may be chosen for its biocompatibility and/or its ability tocontrol immunogenicity of the resultant particle.

In one set of embodiments, the particles are formed by providing asolution comprising one or more polymers, and contacting the solutionwith a polymer nonsolvent to produce the particle. The solution may bemiscible or immiscible with the polymer nonsolvent. For example, awater-miscible liquid such as acetonitrile, tetrahydrofuran, acetone,formamide, dimethylformamide, pyridine, dioxane, or dimethylsulfoxidemay contain the polymers, and particles are formed as the water-miscibleorganic solution is contacted with water, a polymer nonsolvent, e.g., bypouring the water-miscible organic solution into the water at acontrolled rate. The polymer contained within the water-miscible organicsolution, upon contact with the polymer nonsolvent, may then precipitateto form particles such as nanoparticles.

Two liquids are said to be “immiscible” or not miscible, with each otherwhen one is not soluble in the other to a level of at least 10% byweight at ambient temperature and pressure. Typically, an organicsolution (e.g., dichloromethane, chloroform, etc.) and an aqueous liquid(e.g., water, or water containing dissolved salts or other species, cellor biological media, ethanol, etc.) are immiscible with respect to eachother. For example, the first solution may be poured into the secondsolution (at a suitable rate or speed). In some cases, particles such asnanoparticles may be formed as the first solution contacts theimmiscible second liquid, e.g., precipitation of the polymer uponcontact causes the polymer to form nanoparticles while the firstsolution is poured into the second liquid, and in some cases, forexample, when the rate of introduction is carefully controlled and keptat a relatively slow rate, nanoparticles may form. The control of suchparticle formation can be readily optimized by one of ordinary skill inthe art using only routine experimentation.

In some embodiments, the aqueous solution may contain a surfactant. Forexample, the surfactant may be a non-ionic surfactant, an ionicsurfactant, or a mixture thereof. In some instances, the non-ionicsurfactant may have a hydrophilic-lipophilic-balance (HLB) greater thanabout 10, in some embodiments greater than about 12, in some embodimentsgreater than about 15, and in some embodiments greater than about 18. Insome embodiments, the HLB may be between about 10 and about 20, in someembodiments between about 12 and about 20, in some embodiments betweenabout 15 and about 20, or in some embodiments between about 18 and about20.

In some embodiments, the non-ionic surfactant may be a polymer. Forexample, in some cases, the non-ionic surfactant may comprise ahydrophilic polymer, such as a polyalkylene oxide (e.g., polyethyleneoxide). In some embodiments, hydrophilic polymer (e.g., polyethyleneoxide) may have a molecular weight between about 2 kDa and about 10 kDa,or in some cases between about 4 kDa and about 6 kDa. In someembodiments, the non-ionic surfactant may comprise a hydrophobicpolymer. For example, the hydrophobic polymer may be, in some instances,poly(lactic acid) or poly(lactic acid)-co-(glycolic acid). In someembodiments, the poly(lactic acid) or the poly(lactic acid)-co-(glycolicacid) may have a molecular weight between about 0.2 kDa and about 1.0kDa, or in some cases between about 0.4 kDa and about 0.8 kDa. Incertain embodiments, the non-ionic surfactant may be a block copolymer,e.g., polyethylene oxide-block-poly(lactic acid) or polyethyleneoxide-block-poly(lactic acid)-co-(glycolic acid).

In some embodiments, the surfactant may be a polyoxyalkylene alkylether. For example, in some cases, the polyoxyalkylene alkyl ether maybe polyoxyethylene (100) stearyl ether (i.e., Brij® 100),polyoxyethylene (20) cetyl ether (i.e., Brij® 58), or polyoxyethylene(23) lauryl ether (i.e., Brij® 35). In some embodiments, thepolyoxyalkylene alkyl ether may have a molecular weight of between about1 kDa to about 10 kDa, in some embodiments between about 1 kDa to about8 kDa, in some embodiments between about 1 kDa to about 6 kDa, n someembodiments between about 1 kDa to about 4 kDa, n some embodimentsbetween about 1 kDa to about 2 kDa, in some embodiments between about 2kDa to about 10 kDa, in some embodiments between about 4 kDa to about 10kDa, in some embodiments between about 2 kDa to about 8 kDa, in someembodiments between about 4 kDa to about 6 kDa,

In certain embodiments, the aqueous solution used in formulating thecontemplated nanoparticles (i.e., the aqueous phase) may comprisebetween about 0.01 and about 5 weight percent of the surfactant, in someembodiments between about 0.05 and about 5 weight percent of thesurfactant, in some embodiments between about 0.1 and about 5 weightpercent of the surfactant, in some embodiments between about 0.2 andabout 5 weight percent of the surfactant, in some embodiments betweenabout 1 and about 5 weight percent of the surfactant, in someembodiments between about 2 and about 5 weight percent of thesurfactant, in some embodiments between about 0.01 and about 4 weightpercent of the surfactant in some embodiments between about 0.01 andabout 2 weight percent of the surfactant, in some embodiments betweenabout 0.01 and about 1 weight percent of the surfactant, in someembodiments between about 0.01 and about 0.1 weight percent of thesurfactant, in some embodiments between about 0.05 and about 2 weightpercent of the surfactant, in some embodiments between about 0.05 andabout 1 weight percent of the surfactant, in some embodiments betweenabout 0.05 and about 0.2 weight percent of the surfactant, in someembodiments between about 0.1 and about 2 weight percent of thesurfactant, in some embodiments between about 0.1 and about 1 weightpercent of the surfactant, or in some embodiments between about 1 andabout 2 weight percent of the surfactant.

Properties such as surface functionality, surface charge, size, zetapotential, hydrophobicity, ability to control immunogenicity, and thelike, may be highly controlled using a disclosed process. For instance,a library of particles may be synthesized, and screened to identify theparticles having a particular ratio of polymers that allows theparticles to have a specific density of moieties present on the surfaceof the particle. This allows particles having one or more specificproperties to be prepared, for example, a specific size and a specificsurface density of moieties, without an undue degree of effort.Accordingly, certain embodiments are directed to screening techniquesusing such libraries, as well as any particles identified using suchlibraries. In addition, identification may occur by any suitable method.For instance, the identification may be direct or indirect, or proceedquantitatively or qualitatively.

In another embodiment, a nanoemulsion process is provided, such as theprocess represented in FIGS. 1, 2A, and 2B. For example, a therapeuticagent, a first polymer (for example, a diblock co-polymer such asPLA-PEG or PLGA-PEG and an optional excipient (e.g., selected from thegroup consisting of Methacrylic Acid Copolymer, Type A-NF (i.e.,Eudragit® L 100); Methacrylic Acid Copolymer, Type B-NF (i.e., Eudragit®S 100); and Amino Methacrylate Copolymer-NF (i.e., Eudragit® E PO)), maybe combined with an organic solution to form a first organic phase. Suchfirst phase may include about 1 to about 50% weight solids, about 5 toabout 50% weight solids, about 5 to about 40% weight solids, about 1 toabout 15% weight solids, or about 10 to about 30% weight solids. Thefirst organic phase may be combined with a first aqueous solution,optionally including a surfactant, to form a second phase. The organicsolution can include, for example, toluene, methyl ethyl ketone,acetonitrile, tetrahydrofuran, ethyl acetate, isopropyl alcohol,isopropyl acetate, dimethylformamide, dimethylsulfoxide, methylenechloride, dichloromethane, chloroform, acetone, benzyl alcohol, Tween80, Span 80, or the like, and combinations thereof. In an embodiment,the organic phase may include benzyl alcohol, ethyl acetate, andcombinations thereof. In another embodiment, the organic phase mayinclude benzyl alcohol, ethyl acetate, dimethylformamide,dimethylsulfoxide, and combinations thereof. The second phase can bebetween about 0.01 and 50 weight %, 0.1 and 50 weight %, between about 1and 50 weight %, between about 5 and 40 weight %, or between about 1 and15 weight %, solids. The aqueous solution can be water, optionally incombination with one or more of solutes, e.g., sodium cholate, ethylacetate, benzyl alcohol, or a surfactant selected from the groupconsisting of polyvinyl acetate, polyethylene oxide-block-poly(lacticacid), polyethylene oxide-block-poly(lactic acid-co-glycolic acid), anda Brij® detergent (e.g., Brij® 100, Brij® 58, and/or Brij® 35).

For example, the oil or organic phase may use a solvent that is onlypartially miscible with the nonsolvent (water). Therefore, when mixed ata low enough ratio and/or when using water pre-saturated with theorganic solvents, the oil phase remains liquid. The oil phase may beemulsified into an aqueous solution and, as liquid droplets, shearedinto nanoparticles using, for example, high energy dispersion systems,such as homogenizers or sonicators. The aqueous portion of the emulsion,otherwise known as the “water phase”, may be surfactant solutionconsisting of sodium cholate, polyethylene oxide-block-poly(lacticacid), polyethylene oxide-block-poly(lactic acid-co-glycolic acid),and/or a Brij detergent and pre-saturated with ethyl acetate and benzylalcohol.

Emulsifying the second phase to form an emulsion phase may be performed,for example, in one or two emulsification steps. For example, a primaryemulsion may be prepared, and then emulsified to form a fine emulsion.The primary emulsion can be formed, for example, using simple mixing, ahigh pressure homogenizer, probe sonicator, stir bar, or a rotor statorhomogenizer. The primary emulsion may be formed into a fine emulsionthrough the use of e.g., probe sonicator or a high pressure homogenizer,e.g., by using 1, 2, 3, or more passes through a homogenizer. Forexample, when a high pressure homogenizer is used, the pressure used maybe about 30 to about 60 psi, about 40 to about 50 psi, about 1000 toabout 8000 psi, about 5000 to about 15000 psi, about 2000 to about 4000psi, about 4000 to about 8000 psi, or about 4000 to about 5000 psi,e.g., about 2000, 2500, 4000 or 5000 psi.

Either solvent evaporation or dilution may be needed to complete theextraction of the solvent and solidify the particles. For better controlover the kinetics of extraction and a more scalable process, a solventdilution via aqueous quench may be used. For example, the emulsion canbe diluted into cold water to a concentration sufficient to dissolve allof the organic solvent to form a quenched phase. In some embodiments,quenching may be performed at least partially at a temperature of about5° C. or less. For example, water used in the quenching may be at atemperature that is less that room temperature (e.g., about 0 to about10° C., or about 0 to about 5° C.).

In some embodiments, not all of the therapeutic agent is encapsulated inthe particles at this stage, and a drug solubilizer is added to thequenched phase to form a solubilized phase. The drug solubilizer may befor example, Tween 80, Tween 20, polyvinyl pyrrolidone, cyclodextran,sodium dodecyl sulfate, sodium cholate, diethylnitrosamine, sodiumacetate, urea, glycerin, propylene glycol, glycofurol,poly(ethylene)glycol, bris(polyoxyethyleneglycolddodecyl ether, sodiumbenzoate, sodium salicylate, or combinations thereof. For example,Tween-80 may be added to the quenched nanoparticle suspension tosolubilize the free drug (i.e., therapeutic agent) and prevent theformation of drug crystals. In some embodiments, a ratio of drugsolubilizer to the therapeutic agent is about 200:1 to about 10:1, or insome embodiments about 100:1 to about 10:1.

The solubilized phase may be filtered to recover the nanoparticles. Forexample, ultrafiltration membranes may be used to concentrate thenanoparticle suspension and substantially eliminate organic solvent,free drug (i.e., unencapsulated therapeutic agent), drug solubilizer,and other processing aids (surfactants). Exemplary filtration may beperformed using a tangential flow filtration system. For example, byusing a membrane with a pore size suitable to retain nanoparticles whileallowing solutes, micelles, and organic solvent to pass, nanoparticlescan be selectively separated. Exemplary membranes with molecular weightcut-offs of about 300-500 kDa (˜5-25 nm) may be used.

Diafiltration may be performed using a constant volume approach, meaningthe diafiltrate (cold deionized water, e.g., about 0 to about 5° C., or0 to about 10° C.) may added to the feed suspension at the same rate asthe filtrate is removed from the suspension. In some embodiments,filtering may include a first filtering using a first temperature ofabout 0 to about 5° C., or 0 to about 10° C., and a second temperatureof about 20 to about 30° C., or 15 to about 35° C. In some embodiments,filtering may include processing about 1 to about 30, in some casesabout 1 to about 15, or in some cases 1 to about 6 diavolumes. Forexample, filtering may include processing about 1 to about 30, or insome cases about 1 to about 6 diavolumes, at about 0 to about 5° C., andprocessing at least one diavolume (e.g., about 1 to about 15, about 1 toabout 3, or about 1 to about 2 diavolumes) at about 20 to about 30° C.In some embodiments, filtering comprises processing different diavolumesat different distinct temperatures.

After purifying and concentrating the nanoparticle suspension, theparticles may be passed through one, two or more sterilizing and/ordepth filters, for example, using ˜0.2 μm depth pre-filter. For example,a sterile filtration step may involve filtering the therapeuticnanoparticles using a filtration train at a controlled rate. In someembodiments, the filtration train may include a depth filter and asterile filter.

In another embodiment of preparing nanoparticles, an organic phase isformed composed of a mixture of therapeutic agent and polymer(homopolymer and co-polymer). The organic phase is mixed with an aqueousphase at approximately a 1:5 ratio (oil phase:aqueous phase) where theaqueous phase is composed of a surfactant and some dissolved solvent.The primary emulsion is formed by the combination of the two phasesunder simple mixing or through the use of a rotor stator homogenizer.The primary emulsion is then formed into a fine emulsion through the useof a high pressure homogenizer. The fine emulsion is then quenched byaddition to deionized water under mixing. In some embodiments, thequench:emulsion ratio may be about 2:1 to about 40:1, or in someembodiments about 5:1 to about 15:1. In some embodiments, thequench:emulsion ratio is approximately 8.5:1. Then a solution of Tween(e.g., Tween 80) is added to the quench to achieve approximately 2%Tween overall. This serves to dissolve free, unencapsulated therapeuticagent. The nanoparticles are then isolated through either centrifugationor ultrafiltration/diafiltration.

It will be appreciated that the amounts of polymer and therapeutic agentthat are used in the preparation of the formulation may differ from afinal formulation. For example, some of the therapeutic agent may notbecome completely incorporated in a nanoparticle and such freetherapeutic agent may be e.g., filtered away. For example, in anembodiment, a first organic solution containing about 11 weight percenttheoretical loading of therapeutic agent in a first organic solution, asecond organic solution containing about 89 weight percent polymer(e.g., the polymer may include PLA-PEG), and an aqueous solution may beused in the preparation of a formulation that results in, e.g., a finalnanoparticle comprising about 2 weight percent therapeutic agent andabout 98 weight percent polymer (where the polymer may include PLA-PEG).Such processes may provide final nanoparticles suitable foradministration to a patient that include about 1 to about 20 percent byweight therapeutic agent, e.g., about 1, about 2, about 3, about 4,about 5, about 8, about 10, or about 15 percent therapeutic agent byweight.

Therapeutic Agents

In one aspect, any agent including, for example, therapeutic agents(e.g. anti-cancer agents or anti-inflammatory agents), diagnostic agents(e.g. contrast agents; radionuclides; and fluorescent, luminescent, andmagnetic moieties), prophylactic agents (e.g. vaccines), and/ornutraceutical agents (e.g. vitamins, minerals, etc.) may be delivered bythe disclosed nanoparticles. Exemplary agents to be delivered inaccordance with the present invention include, but are not limited to,small molecules (e.g., cytotoxic agents or anti-inflammatory agents),nucleic acids (e.g., siRNA, RNAi, and mircoRNA agents), proteins (e.g.antibodies), peptides, lipids, carbohydrates, hormones, metals,radioactive elements and compounds, drugs, vaccines, immunologicalagents, etc., and/or combinations thereof. In some embodiments, theagent to be delivered is an agent useful in the treatment of cancer. Inother embodiments, the agent may be useful for the treatment of aninflammatory disease.

In a particular embodiment, the drug or other payload may be released ina controlled release manner from the particle and allowed to interactlocally with the particular targeting site (e.g., a tumor or inflamedtissue). The term “controlled release” (and variants of that term) asused herein (e.g., in the context of “controlled-release system”) isgenerally meant to encompass release of a substance (e.g., a drug) at aselected site or otherwise controllable in rate, interval, and/oramount. Controlled release encompasses, but is not necessarily limitedto, substantially continuous delivery, patterned delivery (e.g.,intermittent delivery over a period of time that is interrupted byregular or irregular time intervals), and delivery of a bolus of aselected substance (e.g., as a predetermined, discrete amount if asubstance over a relatively short period of time (e.g., a few seconds orminutes)).

The active agent or drug may be a therapeutic agent such as anantineoplastic such as mTor inhibitors (e.g., sirolimus, temsirolimus,or everolimus), vinca alkaloids such as vincristine, a diterpenederivative or a taxane such as paclitaxel (or its derivatives such asDHA-paclitaxel or PG-paclitaxel) or cabazitaxel.

In one set of embodiments, the payload is a drug or a combination ofmore than one drug. Such particles may be useful, for example, inembodiments where a targeting moiety may be used to direct a particlecontaining a drug to a particular localized location within a subject,e.g., to allow localized delivery of the drug to occur. Exemplarytherapeutic agents include chemotherapeutic agents such as doxorubicin(Adriamycin), gemcitabine (Gemzar), daunorubicin, procarbazine,mitomycin, cytarabine, etoposide, methotrexate, venorelbine,5-fluorouracil (5-FU), vinca alkaloids such as vinblastine orvincristine; bleomycin, paclitaxel (taxol), docetaxel (taxotere),cabazitaxel, aldesleukin, asparaginase, busulfan, carboplatin,cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethylcamptothecin (SN38),dacarbazine, S-I capecitabine, ftorafur, 5′deoxyflurouridine, UFT,eniluracil, deoxycytidine, 5-azacytosine, 5-azadeoxycytosine,allopurinol, 2-chloroadenosine, trimetrexate, aminopterin,methylene-10-deazaaminopterin (MDAM), oxaplatin, picoplatin,tetraplatin, satraplatin, platinum-DACH, ormaplatin, JM-216, and analogsthereof, epirubicin, etoposide phosphate, 9-aminocamptothecin,10,11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin, TAS103, vindesine, L-phenylalanine mustard, ifosphamidemefosphamide,perfosfamide, trophosphamide carmustine, semustine, epothilones A-E,tomudex, 6-mercaptopurine, 6-thioguanine, amsacrine, etoposidephosphate, karenitecin, acyclovir, valacyclovir, ganciclovir,amantadine, rimantadine, lamivudine, zidovudine, bevacizumab,trastuzumab, rituximab, 5-Fluorouracil, and combinations thereof.

Non-limiting examples of potentially suitable drugs include anti-canceragents, including, for example, cabazitaxel, mitoxantrone, andmitoxantrone hydrochloride. In another embodiment, the payload may be ananti-cancer drug such as 20-epi-1, 25 dihydroxyvitamin D3, 4-ipomeanol,5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin, aclarubicin,acodazole hydrochloride, acronine, acylfiilvene, adecypenol, adozelesin,aldesleukin, all-tk antagonists, altretamine, ambamustine, ambomycin,ametantrone acetate, amidox, amifostine, aminoglutethimide,aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole,andrographolide, angiogenesis inhibitors, antagonist D, antagonist G,antarelix, anthramycin, anti-dorsalizdng morphogenetic protein-1,antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolinglycinate, apoptosis gene modulators, apoptosis regulators, apurinicacid, ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin,asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine, azetepa,azotomycin, baccatin III derivatives, balanol, batimastat,benzochlorins, benzodepa, benzoylstaurosporine, beta lactam derivatives,beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor,bicalutamide, bisantrene, bisantrene hydrochloride,bisazuidinylspermine, bisnafide, bisnafide dimesylate, bistratene A,bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists, breflate,brequinar sodium, bropirimine, budotitane, busulfan, buthioninesulfoximine, cactinomycin, calcipotriol, calphostin C, calusterone,camptothecin derivatives, canarypox IL-2, capecitabine, caraceraide,cabazitaxel, carbetimer, carboplatin, carboxamide-amino-triazole,carboxyamidotriazole, carest M3, carmustine, earn 700, cartilage derivedinhibitor, carubicin hydrochloride, carzelesin, casein kinaseinhibitors, castanosperrnine, cecropin B, cedefingol, cetrorelix,chlorambucil, chlorins, chloroquinoxaline sulfonamide, cicaprost,cirolemycin, cisplatin, cis-porphyrin, cladribine, clomifene analogs,clotrimazole, collismycin A, collismycin B, combretastatin A4,combretastatin analog, conagenin, crambescidin 816, crisnatol, crisnatolmesylate, cryptophycin 8, cryptophycin A derivatives, curacin A,cyclopentanthraquinones, cyclophosphamide, cycloplatam, cypemycin,cytarabine, cytarabine ocfosfate, cytolytic factor, cytostatin,dacarbazine, dacliximab, dactinomycin, daunorubicin hydrochloride,decitabine, dehydrodidemnin B, deslorelin, dexifosfamide, dexormaplatin,dexrazoxane, dexverapamil, dezaguanine, dezaguanine mesylate,diaziquone, didemnin B, didox, diethyhiorspermine,dihydro-5-azacytidine, dioxamycin, diphenyl spiromustine, docetaxel,docosanol, dolasetron, doxifluridine, doxorubicin, doxorubicinhydrochloride, droloxifene, droloxifene citrate, dromostanolonepropionate, dronabinol, duazomycin, duocannycin SA, ebselen, ecomustine,edatrexate, edelfosine, edrecolomab, eflomithine, eflomithinehydrochloride, elemene, elsarnitrucin, emitefur, enloplatin, enpromate,epipropidine, epirubicin, epirubicin hydrochloride, epristeride,erbulozole, erythrocyte gene therapy vector system, esorubicinhydrochloride, estramustine, estramustine analog, estramustine phosphatesodium, estrogen agonists, estrogen antagonists, etanidazole, etoposide,etoposide phosphate, etoprine, exemestane, fadrozole, fadrozolehydrochloride, fazarabine, fenretinide, filgrastim, finasteride,flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine,fludarabine phosphate, fluorodaunorunicin hydrochloride, fluorouracil,flurocitabine, forfenimex, formestane, fosquidone, fostriecin,fostriecin sodium, fotemustine, gadolinium texaphyrin, gallium nitrate,galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, gemcitabinehydrochloride, glutathione inhibitors, hepsulfam, heregulin,hexamethylene bisacetamide, hydroxyurea, hypericin, ibandronic acid,idarubicin, idarubicin hydrochloride, idoxifene, idramantone,ifosfamide, ihnofosine, ilomastat, imidazoacridones, imiquimod,immunostimulant peptides, insulin-like growth factor-1 receptorinhibitor, interferon agonists, interferon alpha-2A, interferonalpha-2B, interferon alpha-N1, interferon alpha-N3, interferon beta-IA,interferon gamma-IB, interferons, interleukins, iobenguane,iododoxorubicin, iproplatm, irinotecan, irinotecan hydrochloride,iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron,jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,lanreotide acetate, leinamycin, lenograstim, lentinan sulfate,leptolstatin, letrozole, leukemia inhibiting factor, leukocyte alphainterferon, leuprolide acetate, leuprolide/estrogen/progesterone,leuprorelin, levamisole, liarozole, liarozole hydrochloride, linearpolyamine analog, lipophilic disaccharide peptide, lipophilic platinumcompounds, lissoclinamide, lobaplatin, lombricine, lometrexol,lometrexol sodium, lomustine, lonidamine, losoxantrone, losoxantronehydrochloride, lovastatin, loxoribine, lurtotecan, lutetium texaphyrinlysofylline, lytic peptides, maitansine, mannostatin A, marimastat,masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinaseinhibitors, maytansine, mechlorethamine hydrochloride, megestrolacetate, melengestrol acetate, melphalan, menogaril, merbarone,mercaptopurine, meterelin, methioninase, methotrexate, methotrexatesodium, metoclopramide, metoprine, meturedepa, microalgal protein kinaseC uihibitors, MIF inhibitor, mifepristone, miltefosine, mirimostim,mismatched double stranded RNA, mitindomide, mitocarcin, mitocromin,mitogillin, mitoguazone, mitolactol, mitomalcin, mitomycin, mitomycinanalogs, mitonafide, mitosper, mitotane, mitotoxin fibroblast growthfactor-saporin, mitoxantrone, mitoxantrone hydrochloride, mofarotene,molgramostim, monoclonal antibody, human chorionic gonadotrophin,monophosphoryl lipid a/myobacterium cell wall SK, mopidamol, multipledrug resistance gene inhibitor, multiple tumor suppressor 1-basedtherapy, mustard anticancer agent, mycaperoxide B, mycobacterial cellwall extract, mycophenolic acid, myriaporone, n-acetyldinaline,nafarelin, nagrestip, naloxone/pentazocine, napavin, naphterpin,nartograstim, nedaplatin, nemorubicin, neridronic acid, neutralendopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxideantioxidant, nitrullyn, nocodazole, nogalamycin, n-substitutedbenzamides, O6-benzylguanine, octreotide, okicenone, oligonucleotides,onapristone, ondansetron, oracin, oral cytokine inducer, ormaplatin,osaterone, oxaliplatin, oxaunomycin, oxisuran, paclitaxel, paclitaxelanalogs, paclitaxel derivatives, palauamine, palmitoylrhizoxin,pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine,pegaspargase, peldesine, peliomycin, pentamustine, pentosan polysulfatesodium, pentostatin, pentrozole, peplomycin sulfate, perflubron,perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate,phosphatase inhibitors, picibanil, pilocarpine hydrochloride,pipobroman, piposulfan, pirarubicin, piritrexim, piroxantronehydrochloride, placetin A, placetin B, plasminogen activator inhibitor,platinum complex, platinum compounds, platinum-triamine complex,plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine,procarbazine hydrochloride, propyl bis-acridone, prostaglandin J2,prostatic carcinoma antiandrogen, proteasome inhibitors, protein A-basedimmune modulator, protein kinase C inhibitor, protein tyrosinephosphatase inhibitors, purine nucleoside phosphorylase inhibitors,puromycin, puromycin hydrochloride, purpurins, pyrazorurin,pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate,RAF antagonists, raltitrexed, ramosetron, RAS farnesyl proteintransferase inhibitors, RAS inhibitors, RAS-GAP inhibitor, retelliptinedemethylated, rhenium RE 186 etidronate, rhizoxin, riboprine, ribozymes,RH retinarnide, RNAi, rogletimide, rohitukine, romurtide, roquinimex,rubiginone Bl, ruboxyl, safingol, safingol hydrochloride, saintopin,sarcnu, sarcophytol A, sargramostim, SDI1 mimetics, semustine,senescence derived inhibitor 1, sense oligonucleotides, signaltransduction inhibitors, signal transduction modulators, simtrazene,single chain antigen binding protein, sizofiran, sobuzoxane, sodiumborocaptate, sodium phenylacetate, solverol, somatomedin bindingprotein, sonermin, sparfosafe sodium, sparfosic acid, sparsomycin,spicamycin D, spirogermanium hydrochloride, spiromustine, spiroplatin,splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-celldivision inhibitors, stipiamide, streptonigrin, streptozocin,stromelysin inhibitors, sulfinosine, sulofenur, superactive vasoactiveintestinal peptide antagonist, suradista, suramin, swainsonine,synthetic glycosaminoglycans, talisomycin, tallimustine, tamoxifenmethiodide, tauromustine, tazarotene, tecogalan sodium, tegafur,tellurapyrylium, telomerase inhibitors, teloxantrone hydrochloride,temoporfin, temozolomide, teniposide, teroxirone, testolactone,tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide,thiamiprine, thiocoraline, thioguanine, thiotepa, thrombopoietin,thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist,thymotrinan, thyroid stimulating hormone, tiazofurin, tin ethyletiopurpurin, tirapazamine, titanocene dichloride, topotecanhydrochloride, topsentin, toremifene, toremifene citrate, totipotentstem cell factor, translation inhibitors, trestolone acetate, tretinoin,triacetyluridine, triciribine, triciribine phosphate, trimetrexate,trimetrexate glucuronate, triptorelin, tropisetron, tubulozolehydrochloride, turosteride, tyrosine kinase inhibitors, tyrphostins, UBCinhibitors, ubenimex, uracil mustard, uredepa, urogenital sinus-derivedgrowth inhibitory factor, urokinase receptor antagonists, vapreotide,variolin B, velaresol, veramine, verdins, verteporfin, vinblastinesulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidinesulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine orvinorelbine tartrate, vinrosidine sulfate, vinxaltine, vinzolidinesulfate, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb,zinostatin, zinostatin stimalamer, or zorubicin hydrochloride.

Non-limiting examples of potentially suitable drugs also includeanti-inflammatory agents, including, for example, anti-inflammatorysteroids and non-steroidal anti-inflammatory agents (NSAIDs).Non-limiting examples of anti-inflammatory agents include methotrexate,cyclosporine, alclometasone, azathioprine, beclometasone dipropionate,betamethasone dipropionate, budesonide, celecoxib, chloroprednisone,ciclesonide, cortisol, cortisporin, cortivazol, deflazacort,dexamethasone, fludroxycortide, flunisolide, fluocinonide,fluocortolone, fluorometholone, fluticasone, fluticasone furoate,fluticasone propionate, glucocorticoids, hydrocortamate, megestrolacetate, mesalazine, meprednisone, 6-mercaptopurine, methylprednisolone,mometasone furoate, paramethasone, prednisolone, prednisone,prednylidene, pregnadiene, pregnatriene, pregnene, proctosedyl,rimexolone, tetrahydrocorticosterone, tobramycin/dexamethasone,triamcinolone, and ulobetasol.

Pharmaceutical Formulations

Nanoparticles disclosed herein may be combined with pharmaceuticallyacceptable carriers to form a pharmaceutical composition, according toanother aspect. As would be appreciated by one of skill in this art, thecarriers may be chosen based on the route of administration as describedbelow, the location of the target issue, the drug being delivered, thetime course of delivery of the drug, etc.

The pharmaceutical compositions can be administered to a patient by anymeans known in the art including oral and parenteral routes. The term“patient,” as used herein, refers to humans as well as non-humans,including, for example, mammals, birds, reptiles, amphibians, and fish.For instance, the non-humans may be mammals (e.g., a rodent, a mouse, arat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). In certainembodiments parenteral routes are desirable since they avoid contactwith the digestive enzymes that are found in the alimentary canal.According to such embodiments, inventive compositions may beadministered by injection (e.g., intravenous, subcutaneous orintramuscular, intraperitoneal injection), rectally, vaginally,topically (as by powders, creams, ointments, or drops), or by inhalation(as by sprays).

In a particular embodiment, the nanoparticles are administered to asubject in need thereof systemically, e.g., by IV infusion or injection.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Inone embodiment, the inventive conjugate is suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) TWEEN™80. The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, theencapsulated or unencapsulated conjugate is mixed with at least oneinert, pharmaceutically acceptable excipient or carrier such as sodiumcitrate or dicalcium phosphate and/or (a) fillers or extenders such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid, (b)binders such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectantssuch as glycerol, (d) disintegrating agents such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate, (e) solution retarding agents such as paraffin, (0absorption accelerators such as quaternary ammonium compounds, (g)wetting agents such as, for example, cetyl alcohol and glycerolmonostearate, (h) absorbents such as kaolin and bentonite clay, and (i)lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof. Inthe case of capsules, tablets, and pills, the dosage form may alsocomprise buffering agents.

It will be appreciated that the exact dosage of a nanoparticlecontaining a therapeutic agent is chosen by the individual physician inview of the patient to be treated, in general, dosage and administrationare adjusted to provide an effective amount of the therapeutic agentnanoparticle to the patient being treated. As used herein, the“effective amount” of a nanoparticle containing a therapeutic agentrefers to the amount necessary to elicit the desired biologicalresponse. As will be appreciated by those of ordinary skill in this art,the effective amount of a nanoparticle containing a therapeutic agentmay vary depending on such factors as the desired biological endpoint,the drug to be delivered, the target tissue, the route ofadministration, etc. For example, the effective amount of a nanoparticlecontaining a therapeutic agent might be the amount that results in areduction in tumor size by a desired amount over a desired period oftime. Additional factors which may be taken into account include theseverity of the disease state; age, weight and gender of the patientbeing treated; diet, time and frequency of administration; drugcombinations; reaction sensitivities; and tolerance/response to therapy.

The nanoparticles may be formulated in dosage unit form for ease ofadministration and uniformity of dosage. The expression “dosage unitform” as used herein refers to a physically discrete unit ofnanoparticle appropriate for the patient to be treated. It will beunderstood, however, that the total daily usage of the compositions willbe decided by the attending physician within the scope of sound medicaljudgment. For any nanoparticle, the therapeutically effective dose canbe estimated initially either in cell culture assays or in animalmodels, usually mice, rabbits, dogs, or pigs. The animal model is alsoused to achieve a desirable concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. Therapeutic efficacy andtoxicity of nanoparticles can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED₅₀ (thedose is therapeutically effective in 50% of the population) and LD₅₀(the dose is lethal to 50% of the population). The dose ratio of toxicto therapeutic effects is the therapeutic index, and it can be expressedas the ratio, LD₅₀/ED₅₀. Pharmaceutical compositions which exhibit largetherapeutic indices may be useful in some embodiments. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for human use.

In an exemplary embodiment, a pharmaceutical composition is disclosedthat includes a plurality of nanoparticles each comprising a therapeuticagent and a pharmaceutically acceptable excipient.

In an embodiment, compositions disclosed herein may include less thanabout 10 ppm of palladium, or less than about 8 ppm, or less than about6 ppm of palladium. For example, provided here is a composition thatincludes nanoparticles wherein the composition has less than about 10ppm of palladium.

In some embodiments, a composition suitable for freezing iscontemplated, including nanoparticles disclosed herein and a solutionsuitable for freezing, e.g., a sugar such as a mono, di, or polysaccharide, e.g., sucrose and/or a trehalose, and/or a salt and/or acyclodextrin solution is added to the nanoparticle suspension. The sugar(e.g., sucrose or trehalose) may act, e.g., as a cryoprotectant toprevent the particles from aggregating upon freezing. For example,provided herein is a nanoparticle formulation comprising a plurality ofdisclosed nanoparticles, sucrose, an ionic halide, and water; whereinthe nanoparticles/sucrose/water/ionic halide is about3-40%/10-40%/20-95%/0.1-10% (w/w/w/w) or about 5-10%/10-15%/80-90%/1-10%(w/w/w/w). For example, such solution may include nanoparticles asdisclosed herein, about 5% to about 20% by weight sucrose and an ionichalide such as sodium chloride, in a concentration of about 10-100 mM.In another example, provided herein is a nanoparticle formulationcomprising a plurality of disclosed nanoparticles, trehalose,cyclodextrin, and water; wherein thenanoparticles/trehalose/water/cyclodextrin is about3-40%/1-25%/20-95%/1-25% (w/w/w/w) or about 5-10%/1-25%/80-90%/10-15%(w/w/w/w).

For example, a contemplated solution may include nanoparticles asdisclosed herein, about 1% to about 25% by weight of a disaccharide suchas trehalose or sucrose (e.g., about 5% to about 25% trehalose orsucrose, e.g. about 10% trehalose or sucrose, or about 15% trehalose orsucrose, e.g. about 5% sucrose) by weight) and a cyclodextrin such asβ-cyclodextrin, in a concentration of about 1% to about 25% by weight(e.g. about 5% to about 20%, e.g. 10% or about 20% by weight, or about15% to about 20% by weight cyclodextrin). Contemplated formulations mayinclude a plurality of disclosed nanoparticles (e.g. nanoparticleshaving PLA-PEG and an active agent), and about 2% to about 15 wt % (orabout 4% to about 6 wt %, e.g. about 5 wt %) sucrose and about 5 wt % toabout 20% (e.g. about 7% wt percent to about 12 wt %, e.g. about 10 wt%) of a cyclodextrin, e.g., HPbCD).

The present disclosure relates in part to lyophilized pharmaceuticalcompositions that, when reconstituted, have a minimal amount of largeaggregates. Such large aggregates may have a size greater than about 0.5μm, greater than about 1 μm, or greater than about 10 μm, and can beundesirable in a reconstituted solution. Aggregate sizes can be measuredusing a variety of techniques including those indicated in the U.S.Pharmacopeia at 32<788>, hereby incorporated by reference. The testsoutlined in USP 32<788> include a light obscuration particle count test,microscopic particle count test, laser diffraction, and single particleoptical sensing. In one embodiment, the particle size in a given sampleis measured using laser diffraction and/or single particle opticalsensing.

The USP 32<788> by light obscuration particle count test sets forthguidelines for sampling particle sizes in a suspension. For solutionswith less than or equal to 100 mL, the preparation complies with thetest if the average number of particles present does not exceed 6000 percontainer that are ≥10 μm and 600 per container that are >25 μm.

As outlined in USP 32<788>, the microscopic particle count test setsforth guidelines for determining particle amounts using a binocularmicroscope adjusted to 100±10× magnification having an ocularmicrometer. An ocular micrometer is a circular diameter graticule thatconsists of a circle divided into quadrants with black reference circlesdenoting 10 μm and 25 μm when viewed at 100× magnification. A linearscale is provided below the graticule. The number of particles withreference to 10 μm and 25 μm are visually tallied. For solutions withless than or equal to 100 mL, the preparation complies with the test ifthe average number of particles present does not exceed 3000 percontainer that are ≥10 μm and 300 per container that are ≥25 μm.

In some embodiments, a 10 mL aqueous sample of a disclosed compositionupon reconstitution comprises less than 600 particles per ml having asize greater than or equal to 10 microns; and/or less than 60 particlesper ml having a size greater than or equal to 25 microns.

Dynamic light scattering (DLS) may be used to measure particle size, butit relies on Brownian motion so the technique may not detect some largerparticles. Laser diffraction relies on differences in the index ofrefraction between the particle and the suspension media. The techniqueis capable of detecting particles at the sub-micron to millimeter range.Relatively small (e.g., about 1-5 weight %) amounts of larger particlescan be determined in nanoparticle suspensions. Single particle opticalsensing (SPOS) uses light obscuration of dilute suspensions to countindividual particles of about 0.5 μm. By knowing the particleconcentration of the measured sample, the weight percentage ofaggregates or the aggregate concentration (particles/mL) can becalculated.

Formation of aggregates can occur during lyophilization due to thedehydration of the surface of the particles. This dehydration can beavoided by using lyoprotectants, such as disaccharides, in thesuspension before lyophilization. Suitable disaccharides includesucrose, lactulose, lactose, maltose, trehalose, or cellobiose, and/ormixtures thereof. Other contemplated disaccharides include kojibiose,nigerose, isomaltose, (β,β-trehalose, α,β-trehalose, sophorose,laminaribiose, gentiobiose, turanose, maltulose, palatinose,gentiobiulose, mannobiase, melibiose, melibiulose, rutinose, rutinulose,and xylobiose. Reconstitution shows equivalent DLS size distributionswhen compared to the starting suspension. However, laser diffraction candetect particles of >10 μm in size in some reconstituted solutions.Further, SPOS also may detect >10 μm sized particles at a concentrationabove that of the FDA guidelines (10⁴-10⁵ particles/mL for >10 μmparticles).

In some embodiments, one or more ionic halide salts may be used as anadditional lyoprotectant to a sugar, such as sucrose, trehalose ormixtures thereof. Sugars may include disaccharides, monosaccharides,trisaccharides, and/or polysaccharides, and may include otherexcipients, e.g. glycerol and/or surfactants. Optionally, a cyclodextrinmay be included as an additional lyoprotectant. The cyclodextrin may beadded in place of the ionic halide salt. Alternatively, the cyclodextrinmay be added in addition to the ionic halide salt.

Suitable ionic halide salts may include sodium chloride, calciumchloride, zinc chloride, or mixtures thereof. Additional suitable ionichalide salts include potassium chloride, magnesium chloride, ammoniumchloride, sodium bromide, calcium bromide, zinc bromide, potassiumbromide, magnesium bromide, ammonium bromide, sodium iodide, calciumiodide, zinc iodide, potassium iodide, magnesium iodide, or ammoniumiodide, and/or mixtures thereof. In one embodiment, about 1 to about 15weight percent sucrose may be used with an ionic halide salt. In oneembodiment, the lyophilized pharmaceutical composition may compriseabout 10 to about 100 mM sodium chloride. In another embodiment, thelyophilized pharmaceutical composition may comprise about 100 to about500 mM of divalent ionic chloride salt, such as calcium chloride or zincchloride. In yet another embodiment, the suspension to be lyophilizedmay further comprise a cyclodextrin, for example, about 1 to about 25weight percent of cyclodextrin may be used.

A suitable cyclodextrin may include α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, or mixtures thereof. Exemplary cyclodextrinscontemplated for use in the compositions disclosed herein includehydroxypropyl-β-cyclodextrin (HPbCD), hydroxyethyl-β-cyclodextrin,sulfobutylether-β-cyclodextrin, methyl-β-cyclodextrin,dimethyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, carboxymethylethyl-β-cyclodextrin, diethyl-β-cyclodextrin,tri-O-alkyl-β-cyclodextrin, glucosyl-β-cyclodextrin, andmaltosyl-β-cyclodextrin. In one embodiment, about 1 to about 25 weightpercent trehalose (e.g. about 10% to about 15%, e.g. 5 to about 20% byweight) may be used with cyclodextrin. In one embodiment, thelyophilized pharmaceutical composition may comprise about 1 to about 25weight percent β-cyclodextrin. An exemplary composition may comprisenanoparticles comprising PLA-PEG, an active/therapeutic agent, about 4%to about 6% (e.g. about 5% wt percent) sucrose, and about 8 to about 12weight percent (e.g. about 10 wt. %) HPbCD.

In one aspect, a lyophilized pharmaceutical composition is providedcomprising disclosed nanoparticles, wherein upon reconstitution of thelyophilized pharmaceutical composition at a nanoparticle concentrationof about 50 mg/mL, in less than or about 100 mL of an aqueous medium,the reconstituted composition suitable for parenteral administrationcomprises less than 6000, such as less than 3000, microparticles ofgreater than or equal to 10 microns; and/or less than 600, such as lessthan 300, microparticles of greater than or equal to 25 microns.

The number of microparticles can be determined by means such as the USP32 <788> by light obscuration particle count test, the USP 32<788> bymicroscopic particle count test, laser diffraction, and single particleoptical sensing.

In an aspect, a pharmaceutical composition suitable for parenteral useupon reconstitution is provided comprising a plurality of therapeuticparticles each comprising a copolymer having a hydrophobic polymersegment and a hydrophilic polymer segment; an active agent; a sugar; anda cyclodextrin.

For example, the copolymer may be poly(lactic)acid-block-poly(ethylene)glycol copolymer. Upon reconstitution, a 100 mLaqueous sample may comprise less than 6000 particles having a sizegreater than or equal to 10 microns; and less than 600 particles havinga size greater than or equal to 25 microns.

The step of adding a disaccharide and an ionic halide salt may compriseadding about 5 to about 15 weight percent sucrose or about 5 to about 20weight percent trehalose (e.g., about 10 to about 20 weight percenttrehalose), and about 10 to about 500 mM ionic halide salt. The ionichalide salt may be selected from sodium chloride, calcium chloride, andzinc chloride, or mixtures thereof. In an embodiment, about 1 to about25 weight percent cyclodextrin is also added.

In another embodiment, the step of adding a disaccharide and acyclodextrin may comprise adding about 5 to about 15 weight percentsucrose or about 5 to about 20 weight percent trehalose (e.g., about 10to about 20 weight percent trehalose), and about 1 to about 25 weightpercent cyclodextrin. In an embodiment, about 10 to about 15 weightpercent βcyclodextrin is added. The cyclodextrin may be selected fromα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or mixtures thereof.

In another aspect, a method of preventing substantial aggregation ofparticles in a pharmaceutical nanoparticle composition is providedcomprising adding a sugar and a salt to the lyophilized formulation toprevent aggregation of the nanoparticles upon reconstitution. In anembodiment, a cyclodextrin is also added to the lyophilized formulation.In yet another aspect, a method of preventing substantial aggregation ofparticles in a pharmaceutical nanoparticle composition is providedcomprising adding a sugar and a cyclodextrin to the lyophilizedformulation to prevent aggregation of the nanoparticles uponreconstitution.

A contemplated lyophilized composition may have a therapeutic particleconcentration of greater than about 40 mg/mL. The formulation suitablefor parenteral administration may have less than about 600 particleshaving a size greater than 10 microns in a 10 mL dose. Lyophilizing maycomprise freezing the composition at a temperature of greater than about−40° C., or e.g. less than about −30° C., forming a frozen composition;and drying the frozen composition to form the lyophilized composition.The step of drying may occur at about 50 mTorr at a temperature of about−25 to about −34° C., or about −30 to about −34° C.

In an embodiment, provided herein is a pharmaceutical aqueous suspensioncomprising a plurality of nanoparticles, for example, as disclosedherein, having a glass transition temperature between about 37° C. andabout 50° C., or about 37° C. and about 39° C. in said suspension.

Methods of Treatment

In some embodiments, contemplated nanoparticles may be used to treat,alleviate, ameliorate, relieve, delay onset of, inhibit progression of,reduce severity of, and/or reduce incidence of one or more symptoms orfeatures of a disease, disorder, and/or condition. In some embodiments,contemplated nanoparticles may be used to treat solid tumors, e.g.,cancer and/or cancer cells.

The term “cancer” includes pre-malignant as well as malignant cancers.Cancers include, but are not limited to, blood (e.g., leukemia, chronicmyelogenous leukemia, chronic myelomonocytic leukemia, acutelymphoblastic leukemia, Philadelphia chromosome positive acutelymphoblastic leukemia, mantle cell lymphoma, non-Hodgkin's lymphoma,Hodgkin's lymphoma), prostate, gastric cancer, colorectal cancer, skincancer, e.g., melanomas or basal cell carcinomas, lung cancer (e.g.,non-small cell lung cancer), breast cancer, cancers of the head andneck, bronchus cancer, pancreatic cancer, urinary bladder cancer, brainor central nervous system cancer, peripheral nervous system cancer,esophageal cancer, cancer of the oral cavity or pharynx, liver cancer(e.g., hepatocellular carcinoma), kidney cancer (e.g., renal cellcarcinoma, acute nephroblastoma), testicular cancer, biliary tractcancer, small bowel or appendix cancer, gastrointestinal stromal tumor,salivary gland cancer, thyroid gland cancer, adrenal gland cancer,osteosarcoma, chondrosarcoma, cancer of hematological tissues, and thelike. “Cancer cells” can be in the form of a tumor (i.e., a solidtumor), exist alone within a subject (e.g., leukemia cells), or be celllines derived from a cancer.

Cancer can be associated with a variety of physical symptoms. Symptomsof cancer generally depend on the type and location of the tumor. Forexample, lung cancer can cause coughing, shortness of breath, and chestpain, while colon cancer often causes diarrhea, constipation, and bloodin the stool. However, to give but a few examples, the followingsymptoms are often generally associated with many cancers: fever,chills, night sweats, cough, dyspnea, weight loss, loss of appetite,anorexia, nausea, vomiting, diarrhea, anemia, jaundice, hepatomegaly,hemoptysis, fatigue, malaise, cognitive dysfunction, depression,hormonal disturbances, neutropenia, pain, non-healing sores, enlargedlymph nodes, peripheral neuropathy, and sexual dysfunction.

In one aspect, a method for the treatment of cancer is provided. In someembodiments, the treatment of cancer comprises administering atherapeutically effective amount of inventive particles to a subject inneed thereof, in such amounts and for such time as is necessary toachieve the desired result. In certain embodiments, a “therapeuticallyeffective amount” of a contemplated nanoparticle is that amounteffective for treating, alleviating, ameliorating, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of cancer.

In one aspect, a method for administering inventive compositions to asubject suffering from cancer is provided. In some embodiments,particles may be administered to a subject in such amounts and for suchtime as is necessary to achieve the desired result (i.e., treatment ofcancer). In certain embodiments, a “therapeutically effective amount” ofan inventive targeted particle is that amount effective for treating,alleviating, ameliorating, relieving, delaying onset of, inhibitingprogression of, reducing severity of, and/or reducing incidence of oneor more symptoms or features of cancer.

Inventive therapeutic protocols involve administering a therapeuticallyeffective amount of an inventive targeted particle to a healthyindividual (i.e., a subject who does not display any symptoms of cancerand/or who has not been diagnosed with cancer). For example, healthyindividuals may be “immunized” with an inventive targeted particle priorto development of cancer and/or onset of symptoms of cancer; at riskindividuals (e.g., patients who have a family history of cancer;patients carrying one or more genetic mutations associated withdevelopment of cancer; patients having a genetic polymorphism associatedwith development of cancer; patients infected by a virus associated withdevelopment of cancer; patients with habits and/or lifestyles associatedwith development of cancer; etc.) can be treated substantiallycontemporaneously with (e.g., within 48 hours, within 24 hours, orwithin 12 hours of) the onset of symptoms of cancer. Of courseindividuals known to have cancer may receive inventive treatment at anytime.

In other embodiments, disclosed nanoparticles can be used to inhibit thegrowth of cancer cells, e.g., lung or colon cancer cells. As usedherein, the term “inhibits growth of cancer cells” or “inhibiting growthof cancer cells” refers to any slowing of the rate of cancer cellproliferation and/or migration, arrest of cancer cell proliferationand/or migration, or killing of cancer cells, such that the rate ofcancer cell growth is reduced in comparison with the observed orpredicted rate of growth of an untreated control cancer cell. The term“inhibits growth” can also refer to a reduction in size or disappearanceof a cancer cell or tumor, as well as to a reduction in its metastaticpotential. Preferably, such an inhibition at the cellular level mayreduce the size, deter the growth, reduce the aggressiveness, or preventor inhibit metastasis of a cancer in a patient. Those skilled in the artcan readily determine, by any of a variety of suitable indicia, whethercancer cell growth is inhibited.

Inhibition of cancer cell growth may be evidenced, for example, byarrest of cancer cells in a particular phase of the cell cycle, e.g.,arrest at the G2/M phase of the cell cycle. Inhibition of cancer cellgrowth can also be evidenced by direct or indirect measurement of cancercell or tumor size. In human cancer patients, such measurementsgenerally are made using well known imaging methods such as magneticresonance imaging, computerized axial tomography and X-rays. Cancer cellgrowth can also be determined indirectly, such as by determining thelevels of circulating carcinoembryonic antigen, prostate specificantigen or other cancer-specific antigens that are correlated withcancer cell growth. Inhibition of cancer growth is also generallycorrelated with prolonged survival and/or increased health andwell-being of the subject.

Also provided herein are methods of administering to a patient ananoparticle disclosed herein including an active agent, wherein, uponadministration to a patient, such nanoparticles substantially reducesthe volume of distribution and/or substantially reduces free C_(max), ascompared to administration of the agent alone (i.e., not as a disclosednanoparticle).

Also provided herein are methods of treating an inflammatory disease ina patient in need thereof. The method comprises administering to thepatient a therapeutically effective amount of the inventivenanoparticles. In some embodiments, the inflammatory disease may beinflammatory bowel disease, such as Crohn's disease, ulcerative colitis,collagenous colitis, lymphocytic colitis, ischemic colitis, diversioncolitis, Behçet's disease, or indeterminate colitis. In otherembodiments, a method of treating irritable bowel syndrome in a patientin need thereof is provided. The method comprises administering to thepatient a therapeutically effective amount of inventive nanoparticles.In some embodiments, the nanoparticles may contain a therapeutic agent.For example, in certain embodiments, the therapeutic agent may be ananti-inflammatory agent, such as described above.

Examples

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodiments,and are not intended to limit the invention in any way.

Example 1: Patupilone Nanoparticle Formulations Containing a EudragitExcipient

Therapeutic nanoparticles were produced using the following formulation:

-   -   25% (w/w) theoretical drug;    -   75% (w/w) Polymer (80% 16/5 PLA-PEG (16 kDa PLA, 5 kDa PEG) and        20% Eudragit polymer);    -   % Total Solids=10%; and    -   Solvents: 20% benzyl alcohol/water (92.5% benzyl alcohol, 7.5%        water (w/w)), 80% ethyl acetate (w/w).

For a 1 gram batch size, 125 mg of drug were dissolved in benzyl alcoholcontaining 7.5 wt. % water to form a drug solution. 16/5 PLA-PEG (300mg) was dissolved in ethyl acetate to form a PLA-PEG solution. Eudragitpolymer (75 mg) was dissolved in either drug solution or PLA-PEGsolution. Drug solution and polymer solution were mixed immediatelybefore formulation of the nanoparticles.

Therapeutic nanoparticles are produced as follows. In order to prepare adrug/polymer solution, 125 mg of patupilone were added to a 20 mL glassvial along with 900 mg of benzyl alcohol containing 7.5% water. Themixture was vortexed until the drug was mostly dissolved, therebyforming the drug solution. To a second 20 mL glass vial was added 300 mgof 16/5 PLA-PEG and 3.6 g of ethyl acetate. The mixture was vortexeduntil the PLA-PEG was mostly dissolved, thereby forming the polymersolution. Eudragit (75 mg) was added to either the drug solution or thepolymer solution and vortexed until most of the Eudragit was dissolved.The polymer solution was then poured into the drug solution and vortexeduntil a clear solution was observed.

An aqueous solution was prepared containing 0.1% Brij 100 dissolved in asolution of 4% benzyl alcohol in water (w/w). Specifically, a 5% Brij100 solution containing 4% benzyl alcohol was prepared by mixing 227.5 gwater, 10 g benzyl alcohol, and 12.5 g Brij 100 in a 500 mL glass bottleon a stir plate until all of the components were dissolved, therebyforming a concentrated aqueous solution. The concentrated aqueoussolution was then cooled to less than 5° C. A diluent composed of 4%benzyl alcohol in water was prepared by mixing 80 g of benzyl alcoholand 1920 g of water in a 500 mL glass bottle on a stir plate untildissolved. The diluent was then cooled to less than 5° C. The aqueoussolution was then prepared by adding 10 g of the concentrated aqueoussolution to 490 g of diluent and mixing on a stir plate.

An emulsion was formed by combining the organic phase into the aqueoussolution at a ratio of 5:1 (aqueous phase:oil phase). The organic phasewas poured into the aqueous solution and homogenized using a handhomogenizer for 15 seconds at room temperature to form a coarseemulsion. The coarse emulsion was subsequently fed through a highpressure homogenizer (110S) by setting the pressure to 45 psi for onepass to form a nanoemulsion.

The nanoemulsion was quenched into cold DI water at <5° C. whilestirring on a stir plate. The ratio of Quench to Emulsion was 10:1. 35%(w/w) Tween 80 in water was then added to the quenched emulsion at aratio of 150:1 (Tween 80:drug).

The nanoparticles were concentrated through tangential flow filtration(TFF) followed by diafiltration to remove solvents, unencapsulated drug,and solubilizer. A quenched emulsion was initially concentrated throughTFF using a 300 KDa Pall cassette (3 membrane) to an approximately 200mL volume. This was followed by diafiltration using approximately 20diavolumes (4 L) of cold DI water. The volume was minimized by adding100 mL of cold water to the vessel and pumping through the membrane forrinsing. Approximately 60 mL of material were collected in a glass vial.

In order to determine the solids concentration of unfiltered finalslurry, a 10 mL volume of final slurry was added to a tared 20 mLscintillation vial and dried under vacuum on lyo/oven. Subsequently, theweight of nanoparticles was determined in the volume of the dried downslurry. Concentrated sucrose (0.666 g/g) is added to the final slurrysample to attain a final concentration of 10% sucrose.

In order to determine the solids concentration of 0.45 μm filtered finalslurry, a portion of the final slurry sample is filtered before theaddition of sucrose using a 0.45 μm syringe filter. A volume of thefiltered sample is then added to a tared 20 mL scintillation vial anddried under vacuum on lyo/oven. The remaining sample of unfiltered finalslurry is frozen with sucrose.

Three Eudragit formulations of Patupilone, lots 281-8-1, 281-80-1R, and281-8-2, were made of under different emulsification conditions aslisted in Table 1 below. Lot 186-101-8 was made without Eudragit polymerunder comparable conditions.

The data in Table 1 show drug load and particle size for allformulations. Compared to nanoparticles (NPs) prepared without usingEudragit, the drug loading of Eudragit NPs showed an increase of morethan threefold, up to 15.18%.

TABLE 1 16/5 Drug PLA- Eudragit theoretical Solid size PEG Lot#excipient loading (%) conc Loading % (nm) Notes 186-101-8 No 15 15% 2.9283.8 0.01% Docusate, 1pass at 10485 psi 281-8-1 Eudragit L100, 25 10%11.57% 145.1 0.1% Brij, 20% of 1pass at 46 psi polymer 281-8-1R EudragitEPO, 25 10% 11.70% 117.2 0.1% Brij, 20% of 1pass at 46 psi polymer281-8-2 Eudragit S100, 25 10% 15.18% 125.8 0.14% Brij, 20% of 2passes atpolymer 46 psi

FIG. 3 shows in vitro release data for the formulations of Table 1.Nearly 100% of the patupilone was released from the NPs after one hourregardless of whether or not the formulation contained Eudragit.Additionally, the Eudragit excipient in the NPs did not change in vitrorelease profiles.

Example 2: Docetaxel Nanoparticle Formulations Containing a EudragitExcipient

Nanoparticles containing docetaxel were prepared similarly as describedabove in Example 1. Table 2 shows NP properties and release rates fordocetaxel nanoparticles.

TABLE 2 Impact of Eudragit, hydrophobic CD, and combination ofEudragit/CD on in vitro release of docetaxel (DTXL) from 16/5 PLA-PEGNPs. In vitro release as a function of time (% Size docetaxel) Lot#Excipient Loading (nm) 0 h 1 h 2 h 4 h 24 h 287-07-5 Control: 8.66 13415 50 61 71 96 DTXL 287-07-3 DTXL: 9.15 134 9 28 34 40 55 10% EudragitS100 284-30-3 DTXL: 9.23 134 13 28 33 36 51 18% Eudragit S100 287-38-2DTXL: 8.10 135 43 62 66 72 92 28% Eudragit S100 287-07-1 DTXL: 8.59 1376 15 17 20 27 10% Eudragit S100, 25% CD 287-07-4 DTXL: 7.56 139 7 22 2630 41 25% CD 284-30-2 DTXL: 7.14 125 6 15 18 21 29 32% CD

FIG. 4 shows in vitro release data for the formulations of Table 2.Addition of Eudragit, CD, or the combination of Eudragit and CD sloweddown the release of docetaxel in comparison to polymer alone. Oneexception was the addition of 28% Eudragit, which showed similar releaseto polymer alone. Formulations containing 10% or 18% Eudragit hadsimilar release profiles. Increasing the amount of CD from 25% to 32%slowed release. The combination of 10% Eudragit/25% CD showed a similarrelease profile as 32% CD alone. The addition of Eudragit, CD, or thecombination of both impacts docetaxel in vitro release. In all cases, itslowed down the release with the exception of the addition of 28%Eudragit, as noted above.

Example 3: Celecoxib Nanoparticle Formulations Containing a EudragitExcipient

Nanoparticles containing celecoxib were prepared similarly as describedabove in Example 1. Table 3 shows NP properties and release rates forcelecoxib nanoparticles.

TABLE 3 Impact of Eudragit, hydrophobic CD, and combination ofEudragit/CD on in vitro release of celecoxib from 50/5 PLA-PEG NPs. Invitro release as a function of Size time (% docetaxel) Lot# ExcipientLoading (nm) 0 h 1 h 2 h 4 h 118-86-8 Control: 50/5 18.3 133 25.4 96.099.4 98.3 PLA-PEG 118-151-1 Control: 50/5 3.48 146.2 20.6 79.0 89.1 95.7PLA-PEG 287-20-3 Celecoxib: 8.72 154 29.1 88.0 94.4 98.7 10% EudragitS100 287-20-1 Celecoxib: 7.77 186 21.8 59.0 64.7 75.6 10% Eudragit S100,25% CD 287-20-5 Celecoxib: 2.38 137 33.7 74.4 79.5 79.0 10% EudragitS100, 25% CD 287-20-7 Celecoxib: 7.14 112 26.1 83.9 89.6 94.4 19% CD284-52-2 Celecoxib: 10.54 221 18.0 78.1 84.0 87.6 19% CD 287-20-4Celecoxib: 7.53 119 28.9 81.5 84.3 92.9 25% CD 284-52-1 Celecoxib: 11.23228 19.5 70.5 78.0 84.5 25% CD 287-20-6 Celecoxib: 6.81 120 26.1 74.681.5 86.0 29% CD

FIG. 5 shows in vitro release data for the formulations of Table 3. Theformulations released between approximately 75-96% of the celecoxibafter 1 hour with the exception of the 287-20-1 formulation (186 nmparticle), which released approximately 60% of the celecoxib after 1hour.

Example 4: Docetaxel Nanoparticle Formulations Containing a PEO-PLAExcipient

Therapeutic nanoparticles were produced using the following formulation:

20% (w/w) theoretical drug;

80% (w/w) Polymer (16/5 PLA-PEG);

% Total Solids=20%;

Solvents: 21% benzyl alcohol, 79% ethyl acetate (w/w);

Surfactant in aqueous phase: PEO(5 kDa)-block-PLA(0.5 kDa), PEO(5kDa)-block-PLA(0.6 kDa), or sodium cholate.

For a 0.5 gram batch size (lots 150-155-1 and 150-155-2), 100 mg of drug400 mg of 16/5 PLA-PEG were used. For a 1 gram batch size (lots150-173-2 and 150-173-3), 200 mg of drug and 800 mg of 16/5 PLA-PEG wereused.

Therapeutic nanoparticles are produced as follows. In order to prepare adrug/polymer solution, 100 mg of docetaxel and 400 mg of polymer wereadded to a 7 mL glass vial along with 2000 mg of benzyl alcohol/ethylacetate mixture (21 wt. %/79 wt. %, respectively). The mixture wasvortexed until the drug and polymer were dissolved.

An aqueous solution was prepared containing 0.1% PEO-PLA dissolved in asolution of 2 wt. % benzyl alcohol and 4 wt. % ethyl acetate in water.Specifically, to a 250 mL bottle was added 0.1 g PEO-PLA and 93.9 g ofDI water and the mixture stirred on a stir plate until dissolved. Tothis solution was added 2 g of benzyl alcohol and 4 g of ethyl acetateand the mixture stirred on a stir plate until dissolved.

An emulsion was formed by combining the organic phase into the aqueoussolution at a ratio of 5:1 (aqueous phase:oil phase). The organic phasewas poured into the aqueous solution and homogenized using a handhomogenizer for 10 seconds at room temperature to form a coarseemulsion. The coarse emulsion was subsequently fed through a highpressure homogenizer (110S) by setting the pressure to 45 psi for onepass to form a nanoemulsion.

The nanoemulsion was quenched into cold DI water at <5° C. whilestirring on a stir plate. The ratio of Quench to Emulsion was 5:1.

The nanoparticles were concentrated through tangential flow filtration(TFF) followed by diafiltration to remove solvents, unencapsulated drug,and solubilizer. A quenched emulsion was initially concentrated throughTFF using a 300 KDa Pall cassette (2 membrane) to an approximately 200mL volume. This was followed by diafiltration using approximately 20diavolumes (4 L) of cold DI water. The volume was minimized by adding100 mL of cold water to the vessel and pumping through the membrane forrinsing. Approximately 50-100 mL of material were collected in a glassvial.

In order to determine the solids concentration of unfiltered finalslurry, a 10 mL volume of final slurry was added to a tared 20 mLscintillation vial and dried under vacuum at 80° C. in a vacuum oven.Subsequently, the weight of nanoparticles was determined in the volumeof the dried down slurry. Concentrated sucrose (0.111 g/g) is added tothe final slurry sample to attain a final concentration of 10% sucrose.

In order to determine the solids concentration of 0.45 μm filtered finalslurry, a portion of the final slurry sample is filtered before theaddition of sucrose using a 0.45 syringe filter. A volume of thefiltered sample is then added to a tared 20 mL scintillation vial anddried at 80° C. in a vacuum oven. The remaining sample of unfilteredfinal slurry is frozen with sucrose.

Table 4 shows the conditions used for formulation nanoparticles. Table 5shows certain properties of the nanoparticles. Table 6 shows the resultsof in vitro release of docetaxel from the nanoparticles. FIG. 6 alsoshows the in vitro release properties of nanoparticles prepared usingPEO-PLA versus SC.

TABLE 4 Formulation conditions. Drug PLA- theoretical Solid pass# @ Lot# PEG Drug loading % conc. Surfactant psi# 150-155-1 16/5 Docetaxel 2020% 0.1% 1@45 psi PEO5k- PLA500 150-155-2 16/5 Docetaxel 20 20% 0.1%1@45 psi PEO5k- PLA600 150-155-5 16/5 Docetaxel 20 20% 0.65% SC 1@45 psi150-173-2 16/5 Docetaxel 20 20% 0.55% SC 1@45 psi 150-173-3 16/5Docetaxel 20 20% 0.525% 1@45 psi SC

TABLE 5 Nanoparticle properties. NP Solids Glass transition Lot #Loading % size (nm) (mg/mL) temperature (Tg, ° C.) 150-155-1 9.39% 104.64.475 42.2 150-155-2 10.71% 128 5.00 40.03 150-155-5 3.54% 86.5 3.57541.73 150-173-2 4.79% 114.2 7.425 NA 150-173-3 4.84% 137.1 6.05 NA

TABLE 6 Cumulative in vitro release (%). 150- Time (hours) 150-155-1155-2 150-173-2 150-173-3 0 9.74 17.99 11.31 12.36 1 40.88 43.00 42.8645.58 2 46.68 48.50 49.94 51.28 4 55.09 55.72 57.59 61.25 24 85.73 82.9578.59 86.24 72 94.28 93.30 84.68 92.60

PEO-PLA block polymers used as surfactants in this study are composed ofa long PEG chain and a short PLA chain. Because the highhydrophilic-lipophilic balance (HLB) value, ˜18, this type of polymer iswater soluble and could be used to stabilize an oil/water (O/W)emulsion. For a nanoemulsion method using PLA-PEG polymer to formulatenanoparticles, this type of surfactant can be advantageous, for example,in terms of pharmaceutical acceptance for residual surfactant, becausePEO-PLA has essentially the same components as PLA-PEG.

Four formulations were prepared using PEO5k-PLA0.5k or PEO5k-PLA0.6k assurfactant (150-155-1, 150-155-2, 150-155-3, and 150-155-4). Twoformulations were also prepared as controls using sodium cholatesurfactant (150-155-5 and 150-155-6). Stable emulsions were formed forall formulations.

For 16/5 PLA-PEG, 9.39% to 10.71% docetaxel was loaded in nanoparticlesusing 0.1% of PEO5k-PLA0.5k (150-155-1) or PEO5k-PLA0.6k (150-155-2)solution as the aqueous phase, with particle sizes of 104.6 nm and 128nm, respectively.

For 47/5 PLA-PEG (i.e., 47 kDa PLA-5 kDa PEG), 14.46% to 16.29%docetaxel was loaded in nanoparticles using 1.37% of PEO5k-PLA0.5k(150-155-3) or 1.17% of PEO5k-PLA0.6k (150-155-4) solution as theaqueous phase, with particle sizes of 208.2 nm and 251.1 nm,respectively. These particles could be optimized by using higherconcentration PEO-PLA aqueous solution.

Comparing to corresponding control, 150-155-1 and 150-155-2 versus150-155-5, 150-155-3, and 150-155-4 versus 150-155-6, the NPs preparedusing PEO-PLA as surfactant have significantly higher drug loadings thanthose using sodium cholate as surfactant.

Collected nanoparticle solids concentrations were comparable to controlsdespite the PEO-PLA lots being prepared on a 0.5-gram scale and thesodium cholate (SC) lots being prepared on a 1-gram scale. The lowestsolids concentration for 150-155-3, 1.75 mg/mL, was due to a largecollection volume, which diluted the final NP concentration.

The glass transition temperatures of all PEO-PLA lots were higher orclose to those of NPs using sodium cholate as surfactant and were wellabove 37° C.

In vitro release profiles of all listed lots using SC or PEGylated PLAsurfactants were similar, with cumulative release of about 10-20% atT=0, about 40-45% at T=1 hour, about 45-50% at T=2 hours, and about55-60% at T=4 hours.

Overall, no significant difference was observed for particles producedusing PEO-PLA or sodium cholate as surfactant with respect to drugloading, yield, particle size, and in vitro release. PEO-PLA assurfactant yields equivalent or improved nanoparticles as compared tosodium cholate.

Additionally, much lower PEO-PLA concentrations (0.1% and <2%) were usedas compared to SC (0.65% and 5%). Inclusion of PEO-PLA in a nanoparticleformulation may be advantageous when formulating nanoparticles from highmolecular weight PLA-PEG polymers, which, in some cases, benefit fromsurfactants with higher emulsification capability to producenanoparticles having smaller diameters.

Further benefits to using PEO-PLA over sodium cholate are (1) same typeof polymer as PLA-PEG, which is well-accepted as an injectablepharmaceutical component, (2) non-ionic neutral molecule, which is inertto most compounds, (3) lower concentration than sodium cholate whenmaking nanoparticles under the same conditions, which potentiallybroadens formulation capability.

Example 5: Docetaxel Nanoparticle Formulations Containing a Brij 100Excipient

Therapeutic nanoparticles were produced using the following formulation:

20% (w/w) theoretical drug;

80% (w/w) Polymer (16/5 PLA-PEG or 47/5 PLA-PEG);

% Total Solids=20%;

Solvents: 21% benzyl alcohol, 79% ethyl acetate (w/w);

Surfactant in aqueous phase: Brij 100, or sodium cholate.

For a 1 gram batch size, 200 mg of drug and 800 mg of 16/5 PLA-PEG or47/5 PLA-PEG were used.

Therapeutic nanoparticles are produced as follows. In order to prepare adrug/polymer solution, 200 mg of docetaxel and 800 mg of polymer wereadded to a 20 mL glass vial along with 4000 mg of benzyl alcohol/ethylacetate mixture (21 wt. %/79 wt. %, respectively). The mixture wasvortexed until the drug and polymer were dissolved.

An aqueous solution was prepared containing 0.1% Brij 100 dissolved in asolution of 2 wt. % benzyl alcohol and 4 wt. % ethyl acetate in water.Specifically, to a 1 L bottle was added 1 g Brij 100 and 939 g of DIwater and the mixture stirred on a stir plate until dissolved. To thissolution was added 20 g of benzyl alcohol and 40 g of ethyl acetate andthe mixture stirred on a stir plate until dissolved.

An emulsion was formed by combining the organic phase into the aqueoussolution at a ratio of 5:1 (aqueous phase:oil phase). The organic phasewas poured into the aqueous solution and homogenized using a handhomogenizer for 10 seconds at room temperature to form a coarseemulsion. The coarse emulsion was subsequently fed through a highpressure homogenizer (110S) by setting the pressure to 45 psi for onepass to form a nanoemulsion.

The nanoemulsion was quenched into cold DI water at <5° C. whilestirring on a stir plate. The ratio of Quench to Emulsion was 5:1.

The nanoparticles were concentrated through tangential flow filtration(TFF) followed by diafiltration to remove solvents, unencapsulated drug,and solubilizer. A quenched emulsion was initially concentrated throughTFF using a 300 KDa Pall cassette (2 membrane) to an approximately 200mL volume. This was followed by diafiltration using approximately 20diavolumes (4 L) of cold DI water. The volume was minimized by adding100 mL of cold water to the vessel and pumping through the membrane forrinsing. Approximately 50-100 mL of material were collected in a glassvial.

In order to determine the solids concentration of unfiltered finalslurry, a 10 mL volume of final slurry was added to a tared 20 mLscintillation vial and dried under vacuum at 80° C. in a vacuum oven.Subsequently, the weight of nanoparticles was determined in the volumeof the dried down slurry. Concentrated sucrose (0.111 g/g) is added tothe final slurry sample to attain a final concentration of 10% sucrose.

In order to determine the solids concentration of 0.45 μm filtered finalslurry, a portion of the final slurry sample is filtered before theaddition of sucrose using a 0.45 μm syringe filter. A volume of thefiltered sample is then added to a tared 20 mL scintillation vial anddried at 80° C. in a vacuum oven. The remaining sample of unfilteredfinal slurry is frozen with sucrose.

Table 7 shows the conditions used for formulation nanoparticles. Table 8shows certain properties of the nanoparticles. Table 9 shows the resultsof in vitro release of docetaxel from the nanoparticles. FIG. 7 alsoshows the in vitro release properties of nanoparticles prepared usingBrij 100 versus SC.

TABLE 7 Formulation conditions. Drug theo- PLA- retical Solid pass# @Lot # PEG Drug loading con Surfactant psi# 150-125-2 16/5 Docetaxel 2020% 0.1% Brij 1@45 psi 150-125-4 47/5 Docetaxel 20 20% 1% Brij 2@45 psi150-125-5 47/5 Docetaxel 20 20% 5% SC 1^(st)@60 psi, 2^(nd)@45 psi

TABLE 8 Nanoparticle properties. size NP Solids Glass transitiontemperature Lot # Loading % (nm) (mg/mL) (Tg, ° C.) 150-125-2 4.84%165.8 6.125 37.86 150-125-4 5.22% 144.3 5.1 43.21 150-125-5 4.27% 139.55.15 42.29

TABLE 9 In vitro release. Time Cumulative release (%) (hours) 150-125-2150-125-4 150-125-5 0 21.3 3.6 6.6 1 49.6 19.2 21.9 2 57.0 22.0 26.5 465.4 25.3 29.3 24 89.8 35.4 36.5 77 — 42.33 48.34

Brij 100 is a non-ionic surfactant, polyoxyethylene (100) stearyl ether,with average Mn˜4,670. Its HLB value is 18.8. It is composed of twoneutral blocks, a lipophilic short chain and a hydrophilic PEG chain.Brij 100 is used in pharmaceuticals as an emulsifier, wetting agent, oroil solubilizer. It is a water-soluble pale yellow solid, and isconsidered a non-toxic, nonirritant, noncarcinogenic compound, with anLD50 (oral, rat) >16 g/kg.

Two formulations were prepared using Brij 100 as surfactant (150-125-2and 150-125-4), and one formulation was prepared as a control usingsodium cholate as surfactant (150-125-5).

For 16/5 PLA-PEG, 4.84% docetaxel was loaded in nanoparticles using 0.1%of Brij solution as aqueous phase, with particle size of 165.8 nm(150-125-2). For 47/5 PLA-PEG, 5.22% docetaxel was loaded innanoparticles using 1% Brij (150-125-4). These results are comparable orslightly higher than that of the control batch using sodium cholate assurfactant (150-125-5). The particle sizes of the two lots were similar,144.3 nm for Brij 100 lot versus 139.5 nm for sodium cholate lot.Collected nanoparticles solid concentrations were also similar: 5.1mg/mL versus 5.15 mg/mL. Glass transition temperatures of the two lotsof nanoparticles are both well above physiological temperature, 37° C.,with less than 1° C. difference (43.21° C. versus 42.29° C.).

Docetaxel release from nanoparticles prepared from 16/5 PLA-PEG shows afaster release than that from nanoparticles of 47/5 PLA-PEG, which iscontrolled mainly by polymer MW. Nanoparticles prepared from 47/5PLA-PEG and either Brij 100 or sodium cholate as surfactant show similarrelease profiles, with a minor fluctuation of <±2% difference.

Overall, no significant difference was observed for nanoparticlesproduced using Brij 100 or sodium cholate as surfactant with respect todrug loading, yield, particle size, and in vitro release profiles. UsingBrij 100 as a surfactant yielded similar nanoparticles as compared tousing sodium cholate as the surfactant. In addition, a much lower Brij100 concentration (1%) was used in comparison to sodium cholate (5%)indicating that the concentration of Brij could be increased ifnecessary. Consequently, Brij 100 could be advantageous to use insteadof sodium cholate when formulating nanoparticles from high MW PLA-PEGpolymers, which benefit from surfactants with higher emulsificationcapability to produce nanoparticles having smaller diameters.

Further benefits to using PEO-PLA over sodium cholate are (1) non-ionicneutral molecule, which is inert to most compounds, (2) lowerconcentration than sodium cholate when making nanoparticles under thesame conditions, which potentially broadens formulation capability, and(3) more economical pricing at $86.8/1 kg for Brij 100 versus $91.3/100g for sodium cholate (Sigma).

Example 6: Celecoxib Nanoparticle Formulations Prepared Using aWater-Miscible Organic Solvent

Therapeutic nanoparticles were produced using the following formulation:

-   -   10% (w/w) theoretical drug;    -   90% (w/w) Polymer (45/5 PLA-PEG);    -   % Total Solids=10%;    -   Solvents: 33-98% 21/79 BA/EA (21/79 BA/EA=21% benzyl alcohol,        79% ethyl acetate (w/w))+DMSO or DMF;

For a 1 gram batch size, 100 mg of drug and 900 mg of 45/5 PLA-PEG wereused.

Therapeutic nanoparticles are produced as follows. In order to prepare adrug solution, 100 mg of celecoxib and 990 mg of dimethylsulfoxide wereadded to a 20 mL glass vial and vortexed until the drug was dissolved.To prepare a polymer solution, 900 mg of PLA-PEG were added to a second20 mL glass vial along with 8010 mg of benzyl alcohol/ethyl acetatemixture (21 wt. %/79 wt. %, respectively) and the mixture vortexed untilthe polymer was dissolved. The drug solution and the polymer solutionwere combined and vortexed prior to formulation of the nanoparticles.

An aqueous solution was prepared containing 0.4% sodium cholatedissolved in a solution of 2 wt. % benzyl alcohol and 4 wt. % ethylacetate in water. Specifically, to a 1 L bottle was added 4 g sodiumcholate and 956 g of DI water and the mixture stirred on a stir plateuntil dissolved. To this solution was added 20 g of benzyl alcohol and40 g of ethyl acetate and the mixture stirred on a stir plate untildissolved.

An emulsion was formed by combining the organic phase into the aqueoussolution at a ratio of 5:1 (aqueous phase:oil phase). The organic phasewas poured into the aqueous solution and homogenized using a handhomogenizer for 10 seconds at room temperature to form a coarseemulsion. The coarse emulsion was subsequently fed through a highpressure homogenizer (110S) by setting the pressure to 25 psi for onepass to form a nanoemulsion.

The nanoemulsion was quenched into cold DI water at <5° C. whilestirring on a stir plate. The ratio of Quench to Emulsion was 5:1.

The nanoparticles were concentrated through tangential flow filtration(TFF) followed by diafiltration to remove solvents, unencapsulated drug,and solubilizer. A quenched emulsion was initially concentrated throughTFF using a 300 KDa Pall cassette (2 membrane) to an approximately 200mL volume. This was followed by diafiltration using approximately 20diavolumes (4 L) of cold DI water. The volume was minimized by adding100 mL of cold water to the vessel and pumping through the membrane forrinsing. Approximately 50-100 mL of material were collected in a glassvial.

In order to determine the solids concentration of unfiltered finalslurry, a 10 mL volume of final slurry was added to a tared 20 mLscintillation vial and dried under vacuum at 80° C. in a vacuum oven.Subsequently, the weight of nanoparticles was determined in the volumeof the dried down slurry. Concentrated sucrose (0.111 g/g) is added tothe final slurry sample to attain a final concentration of 10% sucrose.

In order to determine the solids concentration of 0.45 μm filtered finalslurry, a portion of the final slurry sample is filtered before theaddition of sucrose using a 0.45 μm syringe filter. A volume of thefiltered sample is then added to a tared 20 mL scintillation vial anddried at 80° C. in a vacuum oven. The remaining sample of unfilteredfinal slurry is frozen with sucrose.

Table 10 shows the conditions used for formulation nanoparticles. Table11 shows certain properties of the nanoparticles. Table 12 shows theresults of in vitro release of celecoxib from the nanoparticles. FIG. 8also shows the in vitro release properties of nanoparticles preparedusing DMSO.

TABLE 1 Formulation conditions. Organic Drug phase BA/EA theoreticalSolid % SC, solvent Lot # (wt. %) loading (%) conc. pass# @ psi# BA/EA131-133-6 100 10 10% 0.4%, 1@25 psi only (control) Mixture 131-133-1 9810 10% 0.4%, 1@25 psi of 131-133-2 95 10 10% 0.4%, 1@25 psi (BA/EA)131-133-3 89 10 10% 0.4%, 1@25 psi and 131-133-4 78 10 10% 0.4%, 1@30psi DMSO 131-133-5 50 10 10% 0.4%-0.56%, 5@30 psi-60 psi Mixture131-150-4 98 10 10% 0.4%, 1@25 psi of 131-145-5 89 10 10% 0.4%, 1@25 psi(BA/EA) 131-150-6 50 10 10% 0.5%, 2@45 psi and DMF 131-150-2 33 10 6.9% 1%-2%, @45 psi-60 psi

TABLE 2 Nanoparticle properties. Organic Drug phase loading Loading sizeNP Solids Yield solvent Lot # % efficiency % (nm) (mg/mL) (%) BA/EA131-133-6 4.52 45.2 146.4 7.625 65.2 only (control) Mixture 131-133-14.82 48.2 148.4 7.775 67.5 of 131-133-2 4.57 45.7 144.7 6.725 58.6(BA/EA) 131-133-3 5.86 58.6 156.1 6.725 66.5 and 131-133-4 5.9 59 139.87.525 61.1 DMSO 131-133-5 7.96 79.6 178.9 4.125 34.8 Mixture 131-150-44.52 45.2 145.6 7.275 69.3 of 131-145-5 5.17 51.7 139.9 8.975 66.7(BA/EA) 131-150-6 7.65 76.5 160.5 5.525 54.5 and DMF 131-150-2 6.63 66.3502.7 5.275 41.8

TABLE 3 In vitro release of control batch and batches using (BA/EA)mixture with DMSO. Cumulative release (%) Time 131- 131- (hours)131-133-6 131-133-1 131-133-2 131-133-3 133-4 133-5 0 6.89 4.31 4.336.80 7.14 12.01 1 82.98 74.49 83.28 81.73 87.32 79.29 2 92.42 88.6591.88 87.50 95.24 89.32 4 96.70 93.31 94.41 90.59 96.46 93.26 25 99.4096.68 98.13 96.73 100.60 98.58

Nanoparticle (NP) solids concentration in the range of about 5-8 mg/mLwas observed for all formulations, and NP yields were all above 50%,except two batches with lower (BA/EA) content, lot 131-133-5 with 50%(BA/EA) and lot 131-150-2 with 33% (BA/EA).

Particle sizes were well controlled in the range of about 140-160 nm forall batches with BA/EA content ≥50%. When BA/EA content dropped to 33%for lot 131-150-2, significantly larger particles (502.7 nm) wereobserved, even when using up to 2% sodium cholate and 60 psi pressure.

Drug loadings of all formulations were equal to or higher than thecontrol. These results demonstrate the potential to use these mixturesto improve drug loading. In vitro release profiles from batches using(BA/EA) mixture with DMSO overlay with the release from the controlbatch, lot 131-133-6. Adding water miscible solvents to the organicphase do not affect in vitro release of nanoparticles.

Overall, by adding water miscible solvents, DMSO or DMF, to organicphase at reasonable amount of up to 50%, decent nanoparticles could beprepared using BIND nanoemulsion method without changing in vitrorelease of nanoparticles. The value for this modified solvent phase ispotentially broadening the range of drugs which could be encapsulatedusing the nanoemulsion method. Because DMSO and DMF are generally goodsolvents for most compounds, drugs (insoluble in organic solvents usedin the nanoemulsion method, including benzyl alcohol, ethyl acetate, andmethylene chloride) may easily be dissolved in DMSO or DMF. When mixingDMSO or DMF with a non-solvent for a drug (e.g., BA, EA or CH₂Cl₂), thedrug's solubility could be improved generally, which benefits drugencapsulation by increasing theoretical drug loading. Drugs that couldnot be encapsulated or have demonstrated low encapsulation efficiencypreviously could be potentially encapsulated using these modifiedorganic phase solvents.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications,websites, and other references cited herein are hereby expresslyincorporated herein in their entireties by reference.

1. A therapeutic nanoparticle comprising: about 0.05 to about 30 weightpercent of an excipient selected from the group consisting of apolyanionic polymer and a polycationic polymer; about 0.2 to about 35weight percent of a therapeutic agent; and about 35 to about 99.75weight percent of a biocompatible polymer.
 2. The therapeuticnanoparticle of claim 1, wherein the excipient is a polyanionic polymer.3. The therapeutic nanoparticle of claim 2, wherein the polyanionicpolymer is a copolymer of methacrylic acid and methyl methacrylatesubunits.
 4. The therapeutic nanoparticle of claim 3, wherein the ratioof methacrylic acid to methyl methacrylate subunits is between about1:0.9 to about 1:3.
 5. The therapeutic nanoparticle of claim 1, whereinthe excipient is a polycationic polymer.
 6. The therapeutic nanoparticleof claim 5, wherein the polycationic polymer is a copolymer of alkylmethacrylate and dimethylaminoethylmethacrylate.
 7. The therapeuticnanoparticle of claim 6, wherein the polycationic polymer is a copolymerof dimethylaminoethyl methacrylate, butyl methacrylate, and methylmethacrylate subunits.
 8. The therapeutic nanoparticle of claim 7,wherein the ratio of dimethylaminoethyl methacrylate to butylmethacrylate to methyl methacrylate subunits is about 1:2:1.
 9. Thetherapeutic nanoparticle of claim 1, wherein the excipient has amolecular weight of between about 20 kDa and about 60 kDa.
 10. Thetherapeutic nanoparticle of claim 1, wherein the excipient has amolecular weight of between about 100 kDa and about kDa.
 11. Thetherapeutic nanoparticle of claim 1, wherein the excipient has a glasstransition temperature of between about 40° C. and about 50° C.
 12. Thetherapeutic nanoparticle of claim 1, wherein the excipient has a glasstransition temperature of greater than about 100° C.
 13. The therapeuticnanoparticle of claim 1, comprising about 5 to about 25 weight percentof the excipient.
 14. The therapeutic nanoparticle of claim 1, furthercomprising about 0.05 to about 35 weight percent cyclodextrin.
 15. Thetherapeutic nanoparticle of claim 14, comprising about 15 to about 30weight percent cyclodextrin.
 16. The therapeutic nanoparticle of claim14 wherein the cyclodextrin is selected from the group consisting ofα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and mixtures thereof.17. The therapeutic nanoparticle of claim 1, wherein the therapeuticagent is a chemotherapeutic agent.
 18. The therapeutic nanoparticle ofclaim 17, wherein the chemotherapeutic agent is selected from the groupconsisting of docetaxel, vincristine, vinorelbine, an epothilone,epothilone B, fluorouracil, irinotecan, capecitabine, and oxaliplatin.19. The therapeutic nanoparticle of claim 1, wherein the therapeuticagent is a celecoxib.
 20. The therapeutic nanoparticle of claim 1,comprising about 3 to about 20 weight percent of the therapeutic agent.21-64. (canceled)